Primate T-lymphotropic viruses

ABSTRACT

Disclosed are compositions and methods related to the isolation and identification of the primate T-lymphotropic viruses, HTLV-3 and HTLV-4. The diversity of HTLVs was investigated among central Africans reporting contact with NHP blood and body fluids through hunting, butchering, and keeping primate pets. Herein it is shown that this population is infected with a variety of HTLVs, including two retroviruses; HTLV-4 is the first member of a novel phylogenetic lineage that is distinct from all known HTLVs and STLVs; HTLV-3 falls within the genetic diversity of STLV-3, a group that has not previously been seen in humans. The present disclosure also relates to vectors and vaccines for use in humans against infection and disease. The disclosure further relates to a variety of bioassays and kits for the detection and diagnosis of infection with and diseases caused by HTLV-3 and HTLV-4 and related viruses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of co-pending International ApplicationNo. PCT/US2006/005869, filed Feb. 21, 2006, which in turn claims thebenefit of U.S. Provisional Application No. 60/654,484, filed on Feb.21, 2005, now expired. Both of these prior applications are incorporatedby herein by reference.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

Aspects of this invention were made with United States governmentsupport. Therefore, the government has certain rights in the invention.

FIELD

Disclosed are compositions and methods related to the isolation andidentification of the primate T-lymphotropic viruses, HTLV-3 and HTLV-4.The present disclosure also relates to vectors and vaccines for use inhumans against infection and disease.

BACKGROUND

Primate T-lymphotropic viruses (PTLVs) are diverse deltaretroviruses,composed of 3 distinct species (PTLV-1, -2, -3) which by conventionalnomenclature are named ‘STLV’ (simian T-lymphotropic virus) when foundin non-human primates (NHPs) and ‘HTLV’ (human T-lymphotropic virus)when found in humans, regardless of suspected zoonotic origin (Mahieuxet al. 1998; Salemi et al. 1999; Slattery et al. 1999; Courgnaud et al.2004). Like HIV, HTLV has the potential to cause disease and circulateglobally in humans sexually, from mother-to-child, and by exposure tocontaminated blood from transfusions and intravenous drug use. HTLV-1causes adult T-cell leukemia and HTLV-1-associated myelopathy/tropicalspastic paraperesis (HAM/TSP) and other inflammatory diseases (Gessain &Mahieux 2000) and HTLV-2 has been associated with a neurologic diseasesimilar to HAM/TSP (Araujo & Hall 2004). There has been no evidence todate of STLVs crossing into people occupationally exposed to NHPs inlaboratories and primate centers, as has been documented with otherprimate retroviruses, including simian immunodeficiency virus (SIV)(Khabbaz et al. 1994), simian foamy virus (SFV) (Switzer et al. 2004,Heneine et al. 1998), and simian type D retrovirus (Lerche et al. 2001).Nevertheless, ongoing zoonotic transmission of STLV to widespread humanpopulations naturally exposed to NHPs through hunting or butchering,similar to that recently reported for SFV in African hunters (Wolfe etal. 2004b), would be of particular public health significance due to thetransmissible and pathogenic nature of this group of viruses amonghumans. HTLV outside of the PTLV-1 and PTLV-2 groups has not previouslybeen documented (Busch et al. 2000; VanDamme et al. 1997; Salemi et al.1999; Slattery et al. 1999).

SUMMARY

Disclosed herein are compositions and methods that include the full andpartial nucleic acid sequences of primate T-lymphotropic viruses knownas HTLV-3 and HTLV-4, including viral fragments. These viruses areuseful as reagents for the screening of human populations for theprevalence of such viruses. The disclosed viruses also can serve asvectors in gene therapy because the viruses appear to not be transmittedfrom humans to other humans. Additionally, the disclosed viruses can beused as reagents in pathogenicity studies of these and related viruses.Moreover, the sequences of the primate T-lymphotropic viruses describedherein can be used as probes to detect virus in biological samples.Vectors are disclosed that express the HTLV-3 and HTLV-4 nucleic acidsequences, and include, but are not limited to, prokaryotic, eukaryoticand viral vectors. The disclosed viruses also can be used as liverecombinant virus vaccines. Additionally, the disclosed viruses can beused as replicating viral systems to kill live dividing cells, either invitro or in vivo.

The present disclosure also includes the isolation and characterizationof primate T-lymphotropic viruses, HTLV-3 and HTLV-4, that are believedto have been transmitted from non-human primates to humans at some pointin the past. The primate T-lymphotropic viruses described herein do notappear to be readily transmitted from human to human, and can be used inprotocols for diagnosing primate T-lymphotropic virus infections, and asvectors in gene therapy procedures.

Compositions are provided that include live replicating retroviralvectors, wherein the vector is derived from a primate T-lymphotropicvirus, and wherein the vector also includes a nucleic acid that encodesa primate T-lymphotropic virus peptide, polypeptide, or protein, or afragment of a primate T-lymphotropic virus peptide, polypeptide, orprotein. For example, the vector can be derived from an HTLV-3 or HTLV-4virus. Thus, in one aspect, a composition is provided that includes livereplicating primate T-lymphotropic virus vectors.

Also provided is a method of treating a subject with a condition,wherein the condition is a viral infection, bacterial infection,parasitic infection, proliferative disorder (e.g., cancer), or acondition associated with a genetic or autoimmune disorder. The methodincludes administering to the subject a live replicating viral vector,wherein the immunizing construct is specific for the condition.

Also provided is a method of preventing a condition in a subject,wherein the condition can be a viral infection, bacterial infection,parasitic infection, proliferative disorder, or a condition associatedwith a genetic or autoimmune disorder. The method includes administeringto the subject a live replicating viral vector, wherein theantigen-encoding nucleic acid is specific for the condition. Alsoprovided are methods of using the vectors, isolated viruses, and/orinfectious clones described herein for making viral infection models andusing models to study diseases and potential treatments, as well as themodels themselves.

Also disclosed are methods and compositions for detecting primateT-lymphotropic virus or a protein encoded therein in biological fluids.The disclosure also encompasses antibodies specific for the primateT-lymphotropic virus and antibodies that inhibit the binding ofantibodies specific for the primate T-lymphotropic virus. Theseantibodies can be polyclonal antibodies or monoclonal antibodies, whichalso includes fragments of any type of antibody. Thus, disclosed areantibodies to HTLV-3 or HTLV-4. The antibodies specific for the primateT-lymphotropic virus can be used in diagnostic kits to detect thepresence and quantity of primate T-lymphotropic virus in biologicalfluids or in organs from nonhuman primates for xenotransplantation. Forexample, an HTLV-3 antibody can be used in a diagnostic kit to detectHTLV-3. Antibodies specific for primate T-lymphotropic virus may also beadministered to a human or animal to passively immunize the human oranimal against primate T-lymphotropic virus, thereby reducing infection,for instance after accidental exposure to nonhuman primate bodilyfluids.

Other embodiments of the disclosure are methods and kits for detectingthe presence and quantity of antibodies that bind primate T-lymphotropicvirus, for example in body fluids. Such kits can be used for thedetection of primate T-lymphotropic virus itself, or for the detectionof antibodies to the primate T-lymphotropic virus, and also can be usedto monitor the blood supply for the presence of primate T-lymphotropicvirus. The disclosed kits include, for example, a kit for the detectionof antibodies to HTLV-3 or HTLV-4.

Also included in the disclosure are recombinant live virus vaccines. Thevirus of the present disclosure has areas of its genome that make ituseful for the insertion of exogenous genes. The inserted gene(s) cancode for any protein for which vaccination or gene therapy is desired. Auseful aspect of such recombinant live viruses is that the recombinantHTLV-3 or HTLV-4 does not cause disease in the host organism. Therecombinant live virus vaccines of the present disclosure are a safe wayto provide antigen to the immune system.

Accordingly, provided is a composition comprising a primateT-lymphotropic virus, or a fragment of the viral gene or the encodedprotein. An example of the disclosed primate T-lymphotropic virusincludes, but is not limited to HTLV-3 and HTLV-4. Also provided is amethod of detecting a primate T-lymphotropic virus, such as HTLV-3 orHTLV-4.

Also provided are methods and compositions for detecting the presenceand amount of primate T-lymphotropic virus in a body fluid or organ.Further embodiments are compositions and methods for treating geneticand physiologic disorders using gene therapy techniques that include theprimate T-lymphotropic virus of the present disclosure as a vector fornucleic acid sequences and antisense sequences.

Further embodiments include providing compositions and methods usefulfor manipulating the expression of genes, providing vaccines, providingcompositions and methods for treating viral infections in humans oranimals, providing compositions and methods that are effective intreating genetic diseases, and providing a method of treating microbialinfections in humans or animals. Yet still other embodiments includeproviding for treatments of conditions that are caused in part byrapidly dividing cellular growth, providing live recombinant virusvaccines, and providing diagnostic tools such as antibodies or antigensfor the monitoring of the blood supply or organ and tissue donation forthe presence of primate T-lymphotropic virus.

These and other features and advantages will become apparent after areview of the following detailed description of the disclosedembodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate several embodiments and, togetherwith the description, illustrate the disclosed compositions and methods.

FIG. 1 is a digital image showing the Western blot serological patternof Human T-cell lymphotropic virus (HTLV) infected African hunters. HTLVclassification based on phylogenetic analyses is provided above specimennames. Reactivity to HTLV-specific proteins is indicated on left.

FIG. 2 shows the phylogenetic relationships of (a) primate T-celllymphotropic virus polymerase (a) PTLV pol (662-bp), (b) PTLV env(297-bp), (c) PTLV tax (730-bp), (d) PTLV-3 long terminal repeat (LTR)(398-bp), and (e) PTLV pol-env-tax region (5258-bp) sequences byneighbour joining analysis. Sequences generated in the current study arenoted with boxes. Nonhuman primate taxon codes are provided in theMethods portion of the Examples section of the specification. Supportfor the branching order was determined by 1,000 bootstrap replicates,and only values 60% or greater are shown. Branch lengths areproportional to the evolutionary distance (scale bar) between the taxa.

FIG. 3 shows the phylogenetic relationships of PTLV type 1 LTR (377-bp)sequences by neighbour joining analysis. Sequences generated in thecurrent study are noted with boxes. Nonhuman primate taxon codes areprovided in the Methods portion of the Examples section of thespecification. Support for the branching order was determined by 1,000bootstrap replicates and only values 60% or greater are shown. Branchlengths are proportional to the evolutionary distance (scale bar)between the taxa.

FIG. 4 shows the strategy for PCR-amplifying the entire HTLV-3(2026ND)genome. Small proviral sequences were first amplified in each major generegion and the long terminal repeat (stippled bars) using genericprimers as described in the Methods portion of the Examples section ofthe specification. The complete proviral sequence was then obtained byusing PCR primers located within each major gene region by genomewalking as indicated with arrows and orange bars. The typical HTLV-1genomic organization is provided for reference.

FIG. 5( a) shows the nucleotide sequence of the HTLV-3(2026ND) LTR andpre-gag region (nucleotides 1-755 of SEQ ID NO: 36). The U3-R-U5locations (vertical lines), the approximate cap site (cap), thepolyadenylation signal, TATA box, the predicted splice donor site(sd-LTR), and two 21-bp repeats are indicated. In the R and U5 regions,the predicted Rex core elements and nuclear riboprotein A1 binding sitesare underlined. The pre-gag region and primer binding site (PBS,underlined) are in italics. FIG. 5( b) shows the plot of predicted RNAstem loop secondary structure of HTLV-3(2026ND) LTR region (nucleotides421-464 of SEQ ID NO: 36). Position of the Rex responsive element(RexRE) core is indicated.

FIG. 6 shows the amino acid sequence of HTLV-3 Tax (SEQ ID NO: 50).Shown in boxes are known functional motifs: NLS, nuclear localizationsignal; (CBP)/P300, cAMP response element (CREB) binding protein; NES,nuclear export signal; CR2, C-terminal transcriptional activating domainbinding; PDZ.

FIG. 7 shows the amino acid sequence of a basic leucine zipper (bZIP)transcription factor from HTLV-3 (SEQ ID NO: 84). Arginine rich andleucine zipper regions of the bZIP protein are boxed.

FIG. 8 shows the phylogenetic relationship of HTLV-3(2026ND) to otherPTLVs (a) entire genome sans long terminal repeat (LTR), (b) gag, (c),polymerase (pol), and (d) envelope (env). Sequences generated in thecurrent study are shown in boxes. Support for the branching order wasdetermined by 1,000 bootstrap replicates; only values of 60% or more areshown. Branch lengths are proportional to the evolutionary distance(scale bar) between the taxa.

FIG. 9 shows the estimated divergence dates for the most recent commonancestor of HTLV-3(2026ND) and other PTLVs. Divergence dates areprovided for each major node of a neighbor-joining tree rooted withPTLV-1 as the outgroup; estimates are provided as ranges using ascalibration points 40,000 and 60,000 years ago (YA) as the separation ofthe Melanesisan HTLV-1 (MEL5) sequence from other PTLV-1 strains.Bootstrap analysis of 1000 replicates is shown on the tree branches;only values>60% are shown.

FIG. 10 (which includes four pages) shows the full-length genomicsequence of HTLV-4(1863LE) (SEQ ID NO: 81).

FIG. 11 shows the plot of predicted RNA stem loop secondary structure ofthe HTLV-4(1863LE) LTR region. Position of the Rex responsive element(RexRE) core is indicated (nucleotides 425-466 of SEQ ID NO: 81).

FIG. 12 shows the phylogenetic relationships of PTLV full-length genomicsequences, including full-length genomic HTLV-3 and HTLV-4. Thesefindings confirm the genetic relationships found earlier that were basedon smaller sequences. Four major phylogroups were inferred with veryhigh bootstrap support. Nonhuman primate taxon codes are provided in theMethods portion of the Examples section of the specification. Supportfor the branching order was determined by 1,000 bootstrap replicates andonly values 60% or greater are shown. Branch lengths are proportional tothe evolutionary distance (scale bar) between the taxa.

FIG. 13 shows the estimated divergence dates for the most recent commonancestor of HTLV-3(2026ND), HTLV-4(1863LE) and other PTLVs. Divergencedates are provided for each major node of a neighbor-joining tree rootedwith PTLV-1 as the outgroup; estimates are provided as ranges using ascalibration points 40,000 and 60,000 years ago (YA) as the separation ofthe Melanesisan HTLV-1 (MEL5) sequence from other PTLV-1 strains. Usingthe bovine leukemia virus (BLV) as an outgroup, a substitution rate of8.6×10⁻⁷ to 1.3×10⁻⁶ substitutions/site/year for PTLV was inferred whichis 3 logs lower than that seen in HIV, confirming the genetic stabilityof these deltaretroviruses. Bootstrap analysis of 1,000 replicates isshown on the tree branches; only values>60% are shown.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

I. Terms

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that throughoutthe document, data are provided in a number of different formats, andthat this data, represents endpoints and starting points, and ranges forany combination of the data points. For example, if a particular datapoint “10” and a particular data point 15 are disclosed, it isunderstood that greater than, greater than or equal to, less than, lessthan or equal to, and equal to 10 and 15 are considered disclosed aswell as between 10 and 15.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Primers” are a subset of probes which are capable of supporting sometype of enzymatic manipulation and which can hybridize with a targetnucleic acid such that the enzymatic manipulation can occur. A primercan be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art which do not interfere withthe enzymatic manipulation.

“Probes” are molecules capable of interacting with a target nucleicacid, typically in a sequence specific manner, for example throughhybridization. The hybridization of nucleic acids is well understood inthe art and discussed herein. Typically a probe can be made from anycombination of nucleotides or nucleotide derivatives or analogsavailable in the art.

Depending on context, the term “virus” is understood to include theinfectious viral particle or the nucleic acide contained therein, orboth.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this disclosure pertains. Thereferences disclosed are also individually and specifically incorporatedby reference herein for the material contained in them that is discussedin the sentence in which the reference is relied upon.

II. Compositions

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular HTLV-3 or HTLV-4 or gene of the HTLV-3 orHTLV-4 such as gag, pol, env, LTR, rex, and tax is disclosed anddiscussed and a number of modifications that can be made are discussed,specifically contemplated is each and every combination and permutationof HTLV-3 or HTLV-4 or genes of the HTLV-3 or HTLV-4 such as gag, pol,env, LTR, rex, and tax and the modifications that are possible unlessspecifically indicated to the contrary. Thus, if a class of molecules A,B, and C are disclosed as well as a class of molecules D, E, and F andan example of a combination molecule, A-D is disclosed, then even ifeach is not individually recited each is individually and collectivelycontemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E,and C-F are considered disclosed. Likewise, any subset or combination ofthese is also disclosed. Thus, for example, the sub-group of A-E, B-F,and C-E would be considered disclosed. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed compositions. Thus, if thereare a variety of additional steps that can be performed it is understoodthat each of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods.

Furthermore, although the disclosed nucleic acid sequences arerepresented as DNA sequences, it is understood that the equivalent RNAsequences also are contemplated. For instance, if a DNA sequencecontains a thymine, it is understood that a uracil also can besubstituted.

Disclosed herein are compositions relating to primate T-lymphotropicviruses HTLV-3 (SEQ ID NO: 36) and HTLV-4 (SEQ ID NOs: 53 and 81). It isunderstood and herein contemplated that the compositions of thedisclosure can comprise the entire HTLV-3 or HTLV-4 virus nucleic acidsequence. It is also understood that the disclosed compositions cancomprise proteins of the disclosed primate T-lymphocyte viruses orfragments of the disclosed proteins. For example, specifically disclosedand herein contemplated are compositions comprising SEQ ID NOs: 1, 3, 5,35, 45, 47, 49, 51, and 52, or any combination thereof. Also disclosedare compositions comprising SEQ ID NOs: 2, 4, 6, 59, 61, and 63 or anycombination thereof. Also disclosed are compositions comprising SEQ IDNOs: 37, 40, 44, 46, 48, and 50 or any combination thereof. Alsodisclosed are compositions comprising SEQ ID NOs: 54, 57, 58, 60, and 62or any combination thereof. Also disclosed are compositions comprisingfragments of the disclosed proteins. Thus, for example are compositionscomprising SEQ ID NOs: 38, 39, 41, 42, and 43 or any combinationthereof. Also disclosed are compositions comprising SEQ ID NOs: 55 and56. It is understood and herein contemplated that any of the disclosedproteins can be used in combination with any of the protein fragments inthe compositions disclosed herein. Thus, for example, disclosed hereinare compositions comprising SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44,46, 48, and 50 or any combination thereof. Also disclosed are SEQ IDNOs: 54, 55, 56, 57, 58, 60, and 62 or any combination thereof. SEQ IDNOs 1-6, 35, and 45 can be used for all the molecular biologicaltechniques known to those skilled in the art. Such uses include, but arenot limited to, generation of probes and vectors containing thesequences, antisense sequences derived from such sequences, and proteinssynthesized using the sequences. RNA and other nucleic acid derivativesare contemplated by the present disclosure.

It is understood that there are known viruses in the art that based oncertain genomic or sequence similarity or taxonomically related to theviruses disclosed herein. It is also understood that the known virusesin the art thought related taxonomically do not encode the specificviruses disclosed herein. Thus specifically disclosed and hereincontemplated are isolated primate T-lymphotropic viruses having a polgene that has less than 63.5% identity to the pol gene of HTLV-1,HTLV-2, STLV-2, and STLV-3, for example, HTLV-4. Also disclosed areisolated primate T-lymphotropic viruses having a gag gene that has lessthan 69% identity to the gag gene of HTLV-1, HTLV-2, STLV-2, and STLV-3,for example, HTLV-3. Also disclosed are isolated primate T-lymphotropicviruses having a pol gene that has less than 62% identity to the polgene of HTLV-1, HTLV-2, STLV-2, and less than 86% identity to the polgene of STLV-3, for example, HTLV-3. Similarly, the disclosed virusescan be distinguished based on the genes encoded by the disclosedviruses, and specifically the identity of said genes to thecorresponding genes of known viruses. Thus, specifically disclosed areisolated primate T-lymphotropic viruses having a LTR that has less than41% identity to the LTR of HTLV-1, HTLV-2 and STLV-3. Also disclosed areisolated primate T-lymphotropic viruses having at least 92.8% identityto the nucleic acid SEQ ID NO: 1.

Also disclosed are isolated primate T-lymphotropic virus having at least92.5% identity to the nucleic acid SEQ ID NO: 3. Also disclosed areprimate T-lymphotropic viruses having at least 94.2% identity to thenucleic acid SEQ ID NO: 5. Also disclosed are primate T-lymphotropicviruses having at least 91.5% identity to the nucleic acid SEQ ID NO:35. Also disclosed are isolated primate T-lymphotropic viruses having atleast 92.8% identity to the nucleic acid SEQ ID NO: 1, at least 92.5%identity to the nucleic acid SEQ ID NO: 3, and at least 94.2% identityto the nucleic acid SEQ ID NO: 5.

HTLV-4 is a unique delta primate T-lymphotropic virus that is distinctfrom all known PTLV lineages with 29-34.4% and 18.3-25% nucleotidedivergence in the conserved pol and tax genes, respectively, a range ofdivergence similar to that between PTLV-1, PTLV-2, and PTLV-3. Thisvirus formed a separate phylogenetic lineage with a long branch lengthand significant bootstrap support in both the pol (FIG. 2 a) and tax(FIG. 2 c) trees. Identical topologies were obtained by using maximumlikelihood analysis. Phylogenetic analyses combined with GenBank blastsearches show that this is the only known virus in this group. For thesereasons, this virus, which was designated HTLV-4, qualifies as the firstmember of a group in the deltaretrovirus genus. Following the guidelinesof the International Committee on Taxonomy of Viruses and pending formalclassification, primate T-lymphotropic virus 4 (PTLV-4) was proposed asthe name for this species, and PTLV-4(1863LE) as the prototype strain.Due to the classification of the virus within the family retroviridae,certain sequence similarity is expected to exist with knownretroviruses. It is understood that the known viruses in the art thoughtto be related taxonomically do not encode the specific viruses disclosedherein. Thus, specifically disclosed and herein contemplated areisolated primate T-lymphotropic viruses having at least 71.5% identityto the nucleic acid SEQ ID NO: 2. Also disclosed are isolated primateT-lymphotropic viruses having at least 73.5% identity to the nucleicacid SEQ ID NO: 4. Also disclosed are isolated primate T-lymphotropicviruses having at least 82% identity to the nucleic acid SEQ ID NO: 6.Also disclosed are isolated primate T-lymphotropic viruses having atleast 71.5% identity to the nucleic acid SEQ ID NO: 2, at least 73.5%identity to the nucleic acid SEQ ID NO: 4, and at least 82% identity tothe nucleic acid SEQ ID NO: 6.

Knowing the sequence for HTLV-3 and/or HTLV-4 allows for various uses ofthe virus and viral sequences. The env gene of HTLV-3 and/or HTLV-4 isnecessary for primate T-lymphotropic virus entry into animal cells. Thegenes of the present disclosure are effective in permitting infection ofcells in a human host. Thus, for example, the env gene is used foruptake of foreign DNA by a wide range of human cells. There has longbeen a need for vectors for getting foreign nucleic acids into cells,both in vivo and in vitro. The introduction of foreign or exogenousnucleic acids into cells has been a technological hurdle for many genetherapy applications and has now been solved by the virus and sequencesherein disclosed. The env sequences can be used with any vector known tothose skilled in the art, and with any other genetic sequences ofchoice, to allow for entry of the nucleic acids into the cells.

The recent advent of technology, and advances in the understanding ofthe structure and function of many genes makes it possible toselectively turn off or modify the activity of a given gene. Alterationof gene activity can be accomplished many ways. For example,oligonucleotides that are complementary to certain gene messages orviral sequences, known as “antisense” compounds, have been shown to havean inhibitory effect against viruses. By creating an antisense compoundthat hybridizes with the targeted RNA message of cells or viruses thetranslation of the message into protein can be interrupted or prevented.In this fashion gene activity can be modulated.

The ability to deactivate specific genes provides great therapeuticbenefits. For example, it is possible to fight viral diseases withantisense molecules that seek out and destroy viral gene products. Intissue culture, antisense oligonucleotides have inhibited infections byherpes-viruses, influenza viruses and the human immunodeficiency virusthat causes AIDS. It is also possible to target antisenseoligonucleotides against mutated oncogenes. Antisense technology alsocan be used to regulate growth and development. However, in order forthe gene therapy to work, antisense sequences must be delivered acrosscellular plasma membranes to the cytosol.

Gene activity is also modified using sense DNA in a technique known asgene therapy. Defective genes are replaced or supplemented by theadministration of “good” or normal genes that are not subject to thedefect. Instead of being defective, the gene may have been deleted, thusreplacement therapy would provide a copy of the gene for use by thecell. The administered normal genes can either insert into a chromosomeor may be present as extracellular DNA and can be used to produce normalRNA, leading to production of the normal gene product. In this fashiongene defects and deficiencies in the production of a gene product may becorrected.

Still further gene therapy has the potential to augment the normalgenetic complement of a cell. For example, one way to combat HIV is tointroduce into an infected person's T cells a gene that makes the cellsresistant to HIV infection. This form of gene therapy is sometimescalled “intracellular immunization.” Genetic material such as apolynucleotide sequence may be administered to a mammal in a viralvector to elicit an immune response against the gene product of theadministered nucleic acid sequence. Such gene vaccines elicit an immuneresponse in the following manner. First, the viral vector containing thenucleic acid sequence is administered to a human or animal. Next, theadministered sequence is expressed to form a gene product within thehuman or animal. The gene product inside the human or animal isrecognized as foreign material and the immune system of the human oranimal mounts an immunological response against the gene product. Theviruses disclosed herein can be used as viral vectors to provide theforeign nucleic acid sequences to the intracellular metabolic processes.

Additionally, gene therapy can be used as a method of delivering drugsin vivo. For example, if genes that code for therapeutic compounds canbe delivered to endothelial cells, the gene products would havefacilitated access to the blood stream. Additionally, cells could beinfected with a retroviral vector such as the present disclosurecarrying nucleic acid sequences coding for pharmaceutical agents thatprevent infection from occurring in the retrovirally infected cells.

The primate T-lymphotropic viruses of the present disclosure can also beused a safe and effective vaccine agent. Genetic sequences forimmunogenic proteins from a variety of infectious agents can beincorporated into the primate T-lymphotropic virus RNA. Once inside acell, the gene product is expressed and releases the immunizing peptideto the body's immune system. In another method, the disclosed virusescan be used to immunize the body against cell markers found on cancer ortumor cells. The genetic sequence of the cancer cell marker isincorporated into the primate T-lymphotropic virus RNA, and afterinfection with the virus, the expressed gene product stimulates theimmune system. The subject's immune system is used to remove thecancerous cells, obviating the need for chemotherapeutic methods.

Such treatment with HTLV-3 or HTLV-4 can be used for any condition inwhich rapidly dividing cells provide an aspect of the pathology of thecondition. One such condition is the presence of uncontrolledangiogenesis within the body. Angiogenesis dependent diseases are wellknown in the art and are caused in part by the rapid growth of bloodvessels. Another such condition is cancer or tumor growth. Cancer ortumors include both solid tumors and other types. Infection with thevirus of the present disclosure, which can cause no disease and does notaffect the host systemically, is an improvement over currently knowntreatments that involved systemically administered agents. Suchchemotherapeutic agents kill rapidly dividing cells but also causetrauma to the entire person. The dosages of such chemotherapeutic agentsmust be titered between killing the cancer and killing the subject.

In contrast, the cancer treatments disclosed are not as harmful to thesubject. The virus can either be administered systemically or injectedin situ into the tumor. The infected cells are killed and tumor growthis stopped. The virus may be administered in one treatment or in aseries of treatments.

The HTLV-3 or HTLV-4 of the present disclosure can be recombinantlymodified to be selective for cellular receptors on the tumor to make thevirus even more specifically targeted to just those cells. Additionally,the virus may have altered promoter regions that can be selectivelyactivated to cause a productive infection. The combination of differentlevels of control of the virus, both natural and recombinantly-produced,are contemplated herein. A virus can be made specific for attachment toonly certain types of cellular receptors, for those cells that aredividing, and will only undergo replication if another exogenouspromoter factor is present. Viral infection by two or more individuallydefective viruses, that require factors or promoters supplied by otherprimate T-lymphotropic viruses or any type of virus, can provide formany levels of control of infection or treatment of specific conditions.

The virus may be administered to the host, for cancer treatment, genetherapy or vaccination by any methods known to those skilled in the art.Such methods include but are not limited to injection, inhalation,ingestion, topical administration and implantation. The virus may bekilled or live, depending on the treatment considered.

The antibodies disclosed herein can be used to detect the presence ofthe disclosed viruses or viral particles. These antibodies can be usedin diagnostic or screening kits to assess the presence of the virus.Additionally, the antibodies can be used to screen organs from nonhumanprimates that may be used in humans. For instance, detection of thepresence of a virus that is transmitted from nonhuman primates to humansis crucial in providing virus-free organs for transplantation.

It is believed that the virus of the present disclosure, comprising theisolates from HTLV-3, is the first definitive isolation of anSTLV-3-like primate T-lymphotropic virus from persons exposed tononhuman primates. This belief is supported by the epidemiology data,the PCR and sequencing data and the serology data and the absence ofsuch reports in the literature. It is understood that HIV-1 and HIV-2used to be called HTLV-III and HTLV-IV before it was known they weredifferent types of viruses. Additionally, the virus of the presentdisclosure comprising the isolates from HTLV-4, are a new species in thedelta primate T-lymphotropic viruses.

III. Vectors

Disclosed are live replicating human primate T-lymphotropic virusvectors suitable for human use comprising an immunizing construct,wherein the immunizing construct is inserted in nontranslated regionbetween env and tax/rex. The disclosed immunizing construct can be anantigen-encoding nucleic acid.

Where reference is made to “antigen”-encoding nucleic acid, it isunderstood that in the context of the disclosure antigens encoded by theantigen-encoding nucleic acid can include but are not limited toimmunogenic or non-immunogenic peptides, polypeptides, proteins,enzymes, cytokines. These antigens can be non-human exogenous antigenicsequences from viruses, bacteria, or parasites. The antigens can also beantigenic endogenous human or human derived sequences from a conditionsuch as a cancer. Also, peptides encoded by the antigen-encoding nucleicacid can include non-antigenic sequences for the purposes of genetherapy.

In another embodiment of the present disclosure, sequences of thedisclosed primate T-lymphotropic viruses can be used for other molecularbiological applications. Regions of the gag gene are important inpackaging genetic material. For example, the gag sequence or regions ofthe sequence are incorporated into other vectors and direct thepackaging of the resultant genetic material for the particularapplication desired, such as packaging recombinant sequences to makealtered infectious virions. Regions of the pol gene are known to becritical for the stable integration of foreign/viral DNA into the hostgenome. Vectors comprising the pol gene sequences can be used tointegrate any DNA into a genome. The primate T-lymphotropic virus andsequences of the present disclosure infect human cells, and thus, thesesequences are used with other foreign or exogenous sequences in humansin methods, including, but not limited to, entry into cells, packaging,and insertion into the genome. Additionally, methods of using thedisclosed primate T-lymphotropic virus and other sequences of thepresent disclosure are not limited to human cells, but all cells thatallow for infection or entry of the nucleic acids.

The present disclosure is directed to compositions and methodscomprising new primate T-lymphotropic viruses, HTLV-3 and/or HTLV-4,particularly compositions and methods for the sequences of the viralgenome. The virus was obtained from humans. The new virus of the presentdisclosure is an excellent vector for gene therapy and for vaccinationpurposes. Additionally, the antibodies or other detection methods fordetecting the new virus are important in detecting the presence of thisand related viruses for xenotransplantation. In addition, the disclosedprimate T-lymphotropic viruses can be used as reagents in pathogenicitystudies of these and related viruses. Moreover, the sequences of thedisclosed primate T-lymphotropic viruses can be used as probes to detectvirus in biological samples. Vectors include but are not limited toprokaryotic, eukaryotic and viral vectors.

Many new useful technologies have been developed that use viral vectorsand form the basis of medical therapies. Examples of such technologiesinclude, but are not limited to, gene replacement, antisense genetherapy, in situ drug delivery, treatment of cancer or infectiousagents, and vaccine therapy. However, to be successful, thesetechnologies require an effective means for the delivery of the geneticinformation across cellular membranes.

It is well-known in the art that vaccinations can be used prophylacticlyfor the prevention of infections as well as therapeutically for thetreatment of ongoing conditions. Such infections or conditions can bebut are not limited to viral infections. Thus, also disclosed arevectors, wherein the antigen-encoding nucleic acid is an antigen from avirus. The viral antigen is selected from the group of virusesconsisting of Herpes simplex virus type-1, Herpes simplex virus type-2,Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Humanherpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Variola virus,Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, SARS,Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measlesvirus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus,Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus,Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus,Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus,Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valleyfever virus, West Nile virus, Rift Valley fever virus, Rotavirus A,Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus,Human T-lymphotropic virus type-1, Human T-lymphotropic virus type-2,Primate T-lymphotropic virus, Hantavirus, Rubella virus, SimianImmunodeficiency virus, Human Immunodeficiency virus type-1, HumanImmunodeficiency virus type-2, and Simian Immunodeficiency virus (SIV).Also disclosed are vectors, wherein the antigen-encoding nucleic acid isSIV-GAG. The art is replete with examples of viral antigens whosesequences and methods of obtaining them are well known.

Vaccinations are also known for the prevention of bacterial infections.Additionally, antibiotics are well-known in the art for the treatment ofvarious bacterial infections. Herein contemplated and disclosed arevectors, wherein the antigen-encoding nucleic acid is an antigen from abacterium. The bacterial antigen is selected from the group consistingof M. tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M.avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M.ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides,other Nocardia species, Legionella pneumophila, other Legionellaspecies, Salmonella typhi, other Salmonella species, Shigella species,Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, otherPasteurella species, Actinobacillus pleuropneumoniae, Listeriamonocytogenes, Listeria ivanovii, Brucella abortus, other Brucellaspecies, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydiatrachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsialspecies, Ehrlichia species, Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillusanthracis, Escherichia coli, Vibrio cholerae, Campylobacter species,Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi,other Hemophilus species, Clostridium tetani, other Clostridium species,Yersinia enterolitica, and other Yersinia species. The art is repletewith examples of bacterial antigens whose sequences and methods ofobtaining them are well known.

Vaccinations are also known for the prevention of fungal infections.Additionally, antibiotics are well-known in the art for the treatment ofvarious fungal infections. Herein contemplated and disclosed arevectors, wherein the antigen-encoding nucleic acid is an antigen from afungus. The fungal antigen can be selected from the group consisting ofCandida albicans, Cryptococcus neoformans, Histoplama capsulatum,Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodesbrasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicilliummarneffi, and Alternaria alternata

The vectors of the disclosure are not limited to fungi, bacteria, andviruses. Also disclosed are vectors, wherein the antigen-encodingnucleic acid is an antigen from a parasite. The parasitic antigen can beselected from the group consisting of Toxoplasma gondii, Plasmodiumfalciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodiumspecies, Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, otherLeishmania species, Schistosoma mansoni, other Schistosoma species, andEntamoeba histolytica. The art is replete with examples of parasiticantigens whose sequences and methods of obtaining them are well known.

There are instances wherein it is advantageous to administer the vectorof the disclosure in a pharmaceutical composition that comprises othervaccines. Pharmaceutical compositions comprising multiple vaccines canbe for therapeutic or prophylactic purposes. Examples of suchcompositions include the mumps, measles, rubella (MMR) vaccine, andvaccines against M. tuberculosis, M. bovis, M. bovis strain BCG, BCGsubstrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M.marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardiaasteroides, other Nocardia species, Legionella pneumophila, otherLegionella species, Salmonella typhi, other Salmonella species, Shigellaspecies, Yersinia pestis, Pasteurella haemolytica, Pasteurellamultocida, other Pasteurella species, Actinobacillus pleuropneumoniae,Listeria monocytogenes, Listeria ivanovii, Brucella abortus, otherBrucella species, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydiatrachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsialspecies, Ehrlichia species, Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillusanthracis, Escherichia coli, Vibrio cholerae, Campylobacter species,Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi,other Hemophilus species, Clostridium tetani, other Clostridium species,Yersinia enterolitica, and other Yersinia species. Specificallycontemplated and disclosed are pharmaceutical compositions comprisingthe vector of the disclosure and one or more additional vaccines. Alsodisclosed are instances in which the vector comprises more than oneantigen-encoding nucleic acid. In such a situation, the vector willproduce each antigen encoded in the vector as a separate antigen.

There are instances in which a disclosed vector alone may not besuitable for a given purpose (e.g., a kit designed to screen potentialdrugs for the treatment of a condition, such kit intended for use inlaboratories without the capabilities to transfect a cell-line with thevector). In such cases, cells previously transfected with the vector ofthe disclosure are needed. Thus, also disclosed are cells comprising thedisclosed vectors.

In one embodiment, the antigen-encoding nucleic acid can encode anon-antigenic sequence of DNA. This sequence provides a functional copyof a disrupted, mutated, disregulated or deleted gene. Examples ofnucleic acids encoding proteins that play a role in genetic disordersare known in the literature relating to genetic disorders. Methods ofmaking these cells are described and exemplified herein and in the art.

The ability to detect the presence of a construct can be a desirablefeature of any vector. As such, vectors often contain a marker to showthat the construct of interest has been delivered to the subject (e.g.,in a cell), and once delivered, is being expressed. A marker can takethe form of a gene that is detectable when expressed. Thus, alsodisclosed are vectors further comprising a reporter gene. One example ofa reporter gene is green fluorescence protein (GFP).

IV. Delivery of the Compositions to Cells

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems, such as electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991). Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

V. Nucleic Acid Based Delivery Systems

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes (e.g., as part of recombinant retrovirus or adenovirus;Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transportnucleic acids into the cell without degradation, and include a promoteryielding expression of the gene in the cells into which it is delivered.In some embodiments the vectors are derived from either a virus orspecifically a retrovirus. Viral vectors can include for example, forexample, HTLV-1, HTLV-2, HTLV-3, HTLV-4, Adenovirus, Adeno-associatedvirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronaltrophic virus, Sindbis and other RNA viruses, including these viruseswith the HIV backbone. Also preferred are any viral families which sharethe properties of these viruses which make them suitable for use asvectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, andretroviruses that express the desirable properties of MMLV as a vector.Retroviral vectors are able to carry a larger genetic payload (e.g., atransgene or marker gene) than other viral vectors, and for this reasonare commonly used vectors. However, they are not as useful innon-proliferating cells. Adenovirus vectors are relatively stable andeasy to work with, have high titers, can be delivered in aerosolformulation, and can transfect non-dividing cells. Pox viral vectors arelarge, have several sites for inserting genes, are thermostable, and canbe stored at room temperature. A preferred embodiment is a viral vectorwhich has been engineered so as to suppress the immune response of thehost organism, elicited by the viral antigens. Preferred vectors of thistype will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promoter cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

VI. Retroviral Vectors

Primate T-lymphotropic viruses are retroviruses. A retrovirus is ananimal virus belonging to the virus family of Retroviridae, includingany types, subfamilies, genus, or tropisms. Retroviral vectors, ingeneral, are described by Verma, I. M., Retroviral vectors for genetransfer, In Microbiology-1985, American Society for Microbiology, pp.229-232, Washington, (1985), which is incorporated by reference herein.Examples of methods for using retroviral vectors for gene therapy aredescribed in U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993));the teachings of which are incorporated herein by reference. Althoughthe present primate T-lymphotropic virus vector is unique, the methodsdescribed for using other types of viral vectors can be useful incertain contexts. See for example U.S. Pat. No. 5,646,032, which isincorporated herein for its teaching of those methods.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serves as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for large fragments of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication, be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of many genes depending onthe size of each transcript. It is preferable to include either positiveor negative selectable markers along with other genes in the insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

A packaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

It is also understood that the pX region can be used to construct avector. The pX region is located between the end of env and thebeginning of Tax and contains small ORFs, hence this is another goodregion for insertion of foreign DNA in an HTLV genome based vector.

Disclosed are methods of detecting the expression of the disclosedvectors comprising using a first antibody to the antigen to measureprotein expression in a quantitative or qualitative way, and furthercomprising detecting the first antibody directly via a calorimetricmeasurement produced through the use of a substrate and a conjugatedantibody or indirectly via a first antibody to the antigen, which inturn is bound by a second antibody that is conjugated and will result ina calorimetric measurement when combined with a substrate.

Also disclosed are methods wherein the antigen is detected by placing analiquot of the disclosed vector in a lane on a gel and probing the gelfor the antigen.

Some methods are methods of detecting the expression of the disclosedvector using a fluorescently labeled first antibody specific for theantigen and visualizing the antigen using a flow cytometer, fluorescencemicroscope, or chemiluminescence. In some embodiments, the firstantibody is not fluorescently labeled, but a target for a secondantibody with a fluorescent label.

Also disclosed are methods of detecting the expression of a disclosedvector comprising using cytolytic killing assay to assess activity, andmethods of detecting the vector that further include obtaining a samplefrom a subject comprising a tissue biopsy or removal of blood or bonemarrow.

VII. Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro. In the methods described above which include theadministration and uptake of exogenous DNA into the cells of a subject(e.g., gene transduction or transfection), delivery of the compositionsto cells can be via a variety of mechanisms. As one example, deliverycan be via a liposome, using commercially available liposomepreparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc.,Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) andTRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as otherliposomes developed according to procedures standard in the art. Inaddition, the disclosed nucleic acid or vector can be delivered in vivoby electroporation, the technology for which is available fromGenetronics, Inc. (San Diego, Calif.) as well as by means of aSONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution or suspension (for example,incorporated into microparticles, liposomes, or cells). These may betargeted to a particular cell type via antibodies, receptors, orreceptor ligands. The following references are examples of the use ofthis technology to target specific proteins to tumor tissue (Senter, etal., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D., Br. J.Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703,(1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, etal., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz andMcKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al.,Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be usedfor a variety of other specific cell types. Vehicles such as “stealth”and other antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral integration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

VIII. In Vivo/Ex Vivo Methods

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

IX. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

X. Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273:113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway et al., Gene 18:355-360(1982)). Of course, promoters from the host cell or related species alsoare useful herein. Such preferred promoters are in the LTRs of HTLV.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3′ (Lusky etal., Mol. Cell. Bio. 3:1108 (1983)) to the transcription unit.Furthermore, enhancers can be within an intron (Banerji et al., Cell33:729 (1983)), as well as within the coding sequence itself (Osborne etal., Mol. Cell. Bio. 4:1293 (1984)). They are usually between 10 and 300bp in length, and they function in cis. Enhancers function to increasetranscription from nearby promoters. Enhancers also often containresponse elements that mediate the regulation of transcription.Promoters can also contain response elements that mediate the regulationof transcription. Enhancers often determine the regulation of expressionof a gene. While many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, fetoprotein and insulin), typicallyone will use an enhancer from a eukaryotic cell virus for generalexpression. Preferred examples are the SV40 enhancer on the late side ofthe replication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

The promotor and/or enhancer may be specifically activated, for instanceby light or specific chemical events which trigger their function.Systems can be regulated by reagents such as tetracycline anddexamethasone. There are also ways to enhance viral vector geneexpression by exposure to irradiation, such as gamma irradiation, oralkylating chemotherapy drugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

XI. Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker is a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells that have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern & Berg, J.Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid, (Mulligan & BergScience 209:1422 (1980)) or hygromycin, (Sugden et al., Mol. Cell. Biol.5:410-413 (1985)). The three examples employ bacterial genes undereukaryotic control to convey resistance to the appropriate drug G418 orneomycin (geneticin), xgpt (mycophenolic acid) or hygromycin,respectively. Others include the neomycin analog G418 and puramycin.

XII. Sequence Similarities

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two proteins or nucleic acids, such as genes. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another method of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith & Waterman Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, and Jaegeret al. Methods Enzymol. 183:281-306, 1989, which are herein incorporatedby reference for at least material related to nucleic acid alignment. Itis understood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith &Waterman calculation method, the Needleman & Wunsch calculation method,the Jaeger calculation methods, or any of the other calculation methods.As yet another example, a first sequence has 80 percent homology, asdefined herein, to a second sequence if the first sequence is calculatedto have 80 percent homology to the second sequence using each ofcalculation methods (although, in practice, the different calculationmethods will often result in different calculated homology percentages).

XIII. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode HTLV-3 orHTLV-4 (e.g., SEQ ID NOs: 36, 53, and 81). The disclosed nucleic acidsare made up of, for example, nucleotides, nucleotide analogs, ornucleotide substitutes. Non-limiting examples of these and othermolecules are discussed herein. It is understood that for example, whena vector is expressed in a cell, the expressed mRNA will typically bemade up of A, C, G, and U. Likewise, it is understood that if, forexample, an antisense molecule is introduced into a cell or cellenvironment through for example exogenous delivery, it is advantageousthat the antisense molecule be made up of nucleotide analogs that reducethe degradation of the antisense molecule in the cellular environment.

XIV. Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Anon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate). There are manyvarieties of these types of molecules available in the art and availableherein.

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties. There are many varieties of these typesof molecules available in the art and available herein.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid. There are many varieties of these types of molecules available inthe art and available herein.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553-6556). There are many varieties of thesetypes of molecules available in the art and available herein.

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

XV. Sequences

There are a variety of sequences related to the protein molecules, forexample the protein coding regions gag, pol, env, tax, rex, and protease(pro) genes and noncoding regions such as the LTR of HTLV-3 and HTLV-4,or any of the nucleic acids disclosed herein for making HTLV-3 orHTLV-4, all of which are encoded by nucleic acids or are nucleic acids.The sequences for the human analogs of these genes, as well as otheranalogs, and alleles of these genes, and splice variants and other typesof variants, are available in a variety of protein and gene databases,including GenBank. Those sequences available at the time of filing thisapplication at GenBank are herein incorporated by reference in theirentireties as well as for individual subsequences contained therein.GenBank can be accessed at http://www.ncbi.nih.gov/entrez/query.fcgi.Those of skill in the art understand how to resolve sequencediscrepancies and differences and to adjust the compositions and methodsrelating to a particular sequence to other related sequences. Primersand/or probes can be designed for any given sequence given theinformation disclosed herein and known in the art.

XVI. Primers and Probes

Disclosed are compositions including primers and probes, which arecapable of interacting with the disclosed nucleic acids, such as theHTLV-3 or HTLV-4 as disclosed herein. In certain embodiments the primersare used to support nucleic acid (DNA, RNA, etc.) amplificationreactions. Thus, for example, disclosed herein are primers wherein theprimer comprises SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs:15 and 16, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31and 32, SEQ ID NOs: 69 and 70, SEQ ID NOs: 73 and 74, SEQ ID NOs: 77 and78, SEQ ID NOs: 9 and 10, SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 18,SEQ ID NOs: 25 and 26, SEQ ID NOs: 29 and 30, SEQ ID NOs: 33 and 34, SEQID NOs: 64 and 65, SEQ ID NOs: 71 and 72, SEQ ID NOs: 75 and 76, and SEQID NOs: 79 and 80. Typically the primers will be capable of beingextended in a sequence specific manner. Extension of a primer in asequence specific manner includes any methods wherein the sequenceand/or composition of the nucleic acid molecule to which the primer ishybridized or otherwise associated directs or influences the compositionor sequence of the product produced by the extension of the primer.Extension of the primer in a sequence specific manner thereforeincludes, but is not limited to, PCR, DNA sequencing, DNA extension, DNApolymerization, RNA transcription, or reverse transcription. Techniquesand conditions that amplify the primer in a sequence specific manner arepreferred. In certain embodiments the primers are used for the DNAamplification reactions, such as PCR or direct sequencing. Thus, hereinare disclosed primer pairs used in conjunction with a second nested setof primers pairs. For example, disclosed herein are PCR amplificationmethods comprising a first primer pair and a second primer pair, whereinthe second primer pair is internal to the first primer pair and whereinthe first primer pair is selected from the group consisting of SEQ IDNOs: 7 and 8, SEQ ID NOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs:23 and 24, SEQ ID NOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 69and 70, SEQ ID NOs: 73 and 74, and SEQ ID NOs: 77 and 78, wherein thesecond set of primers is selected from the group consisting of SEQ IDNOs: 9 and 10, SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 18, SEQ ID NOs:25 and 26, SEQ ID NOs: 29 and 30, SEQ ID NOs: 33 and 34, SEQ ID NOs: 71and 72, SEQ ID NOs: 75 and 76, and SEQ ID NOs: 79 and 80. It isunderstood that in certain embodiments the primers can also be extendedusing non-enzymatic techniques, where for example, the nucleotides oroligonucleotides used to extend the primer are modified such that theywill chemically react to extend the primer in a sequence specificmanner. Typically, the disclosed primers hybridize with the disclosednucleic acids or region of the nucleic acids or they hybridize with thecomplement of the nucleic acids or complement of a region of the nucleicacids.

XVII. Functional Nucleic Acids

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, and external guide sequences. Thefunctional nucleic acid molecules can act as effectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of any of the disclosed nucleicacids, such as the pol, tax, env, gag, rex and pro genes and non-codingregions such as the LTR of HTLV-3 and HTLV-4, or the nucleic acids usedfor the generation of HTLV-3 and HTLV-4, or the genomic DNA of any ofthe disclosed viruses, such as HTLV-3 and HTLV-4, or they can interactwith the polypeptide encoded by any of the disclosed nucleic acids, suchas pol, tax, rex, env, gag, or pro genes of HTLV-3 and HTLV-4, or thenucleic acids used for the generation of pol, tax, rex, env, gag, or LTRproteins of HTLV-3 and HTLV-4. Often functional nucleic acids aredesigned to interact with other nucleic acids based on sequence homologybetween the target molecule and the functional nucleic acid molecule. Inother situations, the specific recognition between the functionalnucleic acid molecule and the target molecule is not based on sequencehomology between the functional nucleic acid molecule and the targetmolecule, but rather is based on the formation of tertiary structurethat allows specific recognition to take place.

XVIII. Protein Variants

As discussed herein, there are numerous disclosed variants of the HTLV-3proteins encoded herein, such as gag (SEQ ID NO: 40), pol (SEQ ID NO:44), env (SEQ ID NO: 37), tax (SEQ ID NO: 50), rex (SEQ ID NO: 48),protease (SEQ ID NO: 46), and non-coding regions such as the LTR, andHTLV-4 proteins encoded herein, such as gag, pol (SEQ ID NO: 57), env(SEQ ID NO: 54), tax (SEQ ID NO: 62), rex (SEQ ID NO: 60), protease (SEQID NO: 58) and non-coding regions such as the LTR. In addition, to theknown functional HTLV-3 and HTLV-4 strain variants there are derivativesof the HTLV-3 and HTLV-4 gag, pol, tax, rex, and env, LTR proteins thatalso function in the disclosed methods and compositions. Proteinvariants and derivatives are well understood to those of skill in theart and in can involve amino acid sequence modifications. For example,amino acid sequence modifications typically fall into one or more ofthree classes: substitutional, insertional or deletional variants.Insertions include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Immunogenic fusion protein derivatives, such as thosedescribed in the examples, are made by fusing a polypeptide sufficientlylarge to confer immunogenicity to the target sequence by cross-linkingin vitro or by recombinant cell culture transformed with DNA encodingthe fusion. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, e.g., a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 1 and 2 and are referred to as conservative substitutions.

TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviation alanine Ala Aallosoleucine AIle arginine Arg R asparagine Asn N aspartic acid Asp Dcysteine Cys C glutamic acid Glu E glutamine Gln K glycine Gly Ghistidine His H isolelucine Ile I leucine Leu L lysine Lys Kphenylalanine Phe F proline Pro P pyroglutamic acid Glu serine Ser Sthreonine Thr T tyrosine Tyr Y tryptophan Trp W valine Val V

TABLE 2 Amino Acid Substitutions Original Residue & ExemplaryConservative Substitutions (others are known in the art) Ala, ser Arg,lys, gln Asn, gln, his Asp, glu Cys, ser Gln, asn, lys Glu, asp Gly, proHis, asn, gln Ile, leu, val Leu, ile, val Lys, arg, gln, Met, Leu, ilePhe, met, leu, tyr Ser, thr Thr, ser Trp, tyr Tyr, trp, phe Val, ile,leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, e.g., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue with another, orone polar residue with another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO: 1 sets forth a particular sequence of HTLV-3 polprotein and SEQ ID NO: 2 sets forth a particular sequence of a HTLV-4pol protein. Specifically disclosed are variants of these and otherproteins herein disclosed which have at least, 70% or 75% or 80% or 85%or 90% or 95% homology or any amount of homology in between to thestated sequence. Those of skill in the art readily understand how todetermine the homology of two proteins. For example, the homology can becalculated after aligning the two sequences so that the homology is atits highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70%, 80%, 85%, 90%, 92%, 95%, 97% or morehomology to a particular sequence wherein the variants are conservativemutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, e.g. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. For example,one of the many nucleic acid sequences that can encode the proteinsequence set forth in SEQ ID NO: 44 is set forth in SEQ ID NO: 1. Inaddition, for example, disclosed are conservative derivatives of SEQ IDNO: 44.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent then the amino acids shown in Table 1 and Table2. The opposite stereo isomers of naturally occurring peptides aredisclosed, as well as the stereo isomers of peptide analogs. These aminoacids can readily be incorporated into polypeptide chains by chargingtRNA molecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEngineering Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CHH₂SO—. (These andothers can be found in Spatola in Chemistry and Biochemistry of AminoAcids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983); Spatola, Vega Data (March 1983), Vol. 1, Issue 3,Peptide Backbone Modifications (general review); Morley, Trends PharmSci (1980) pp. 463-468; Hudson et al., Int J Pept Prot Res 14:177-185(1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. Life Sci 38:1243-1249 (1986)(—CHH₂—S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (—CH—CH—,cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980)(—COCH₂—); Jennings-White et al. Tetrahedron Lett 23:2533 (1982)(—COCH₂—); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405(1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett 24:4401-4404(1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982) (—CH₂—S—);each of which is incorporated herein by reference. A particularlypreferred non-peptide linkage is —CH₂NH—. It is understood that peptideanalogs can have more than one atom between the bond atoms, such asb-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

XIX. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can be administered in vivo in apharmaceutically acceptable carrier. By “pharmaceutically acceptable” ismeant a material that is not biologically or otherwise undesirable,e.g., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,including topical intranasal administration or administration byinhalant. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter et al., BioconjugateChem., 2:447-451, (1991); Bagshawe Br. J. Cancer, 60:275-281, (1989);Bagshawe et al., Br. J. Cancer, 58:700-703, (1988); Senter et al.,Bioconjugate Chem., 4:3-9, (1993); Battelli et al., Cancer Immunol.Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,42:2062-2065, (1991)). Vehicles such as “stealth” and other antibodyconjugated liposomes (including lipid mediated drug targeting to coloniccarcinoma), receptor mediated targeting of DNA through cell specificligands, lymphocyte directed tumor targeting, and highly specifictherapeutic retroviral targeting of murine glioma cells in vivo. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Hughes et al., CancerResearch, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica etBiophysica Acta, 1104:179-187, (1992)). In general, receptors areinvolved in pathways of endocytosis, either constitutive or ligandinduced. These receptors cluster in clathrin-coated pits, enter the cellvia clathrin-coated vesicles, pass through an acidified endosome inwhich the receptors are sorted, and then either recycle to the cellsurface, become stored intracellularly, or are degraded in lysosomes.The internalization pathways serve a variety of functions, such asnutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

XX. Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier. Suitablecarriers and their formulations are described in Remington: The Scienceand Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack PublishingCompany, Easton, Pa. 1995. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarrier include, but are not limited to, saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8, and more preferably from about 7 to about 7.5. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable, depending upon, forinstance, the route of administration and concentration of compositionbeing administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

XXI. Therapeutic Uses

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired therapeuticor prophylactic effect. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient, route ofadministration, or whether other drugs are included in the regimen, andcan be determined by one of skill in the art. The dosage can be adjustedby the individual physician in the event of any counterindications.Dosage can vary, and can be administered in one or more doseadministrations daily, for one or several days. Guidance can be found inthe literature for appropriate dosages for given classes ofpharmaceutical products. For example, guidance in selecting appropriatedoses for antibodies can be found in the literature on therapeutic usesof antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al.,eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haberet al., eds., Raven Press, New York (1977) pp. 365-389. A typical dailydosage of the antibody used alone might range from about 1 μg/kg to upto 100 mg/kg of body weight or more per day, depending on the factorsmentioned above.

Following administration of a disclosed composition, such as anantibody, for treating, inhibiting, or preventing an HTLV-3 or HTLV-4infection, the efficacy of the therapeutic antibody can be assessed invarious ways well known to the skilled practitioner. For instance, oneof ordinary skill in the art will understand that a composition, such asan antibody disclosed herein, is efficacious in treating or inhibitingan HTLV-3 or HTLV-4 infection in a subject by observing that thecomposition reduces viral load or prevents a further increase in HTLV-3or HTLV-4 viral load. Techniques used to measure the response of HTLV-3or HTLV-4-infected subject to treatment with an antibody includedetermining whether the treatment partially or completely inhibits theappearance of the virus in the blood or other body fluid.

Other molecules that interact with HTLV-3 or HLV-4 (or the proteinsencoded by those virus nucleic aid sequences) can be used that do nothave a specific pharmaceutical function, but which may be used fortracking changes within cellular chromosomes or for the delivery ofdiagnostic tools, for example can be delivered in ways similar to thosedescribed for the pharmaceutical products. The disclosed compositionsand methods can also be used, for example, as tools to isolate and testnew drug candidates for a variety of primate T-lymphotropic virusrelated diseases.

XXII. Treatment and Prevention Methods

By “treating” is meant an improvement in or abatement of the diseasestate (e.g., viral infection, bacterial infection, parasitic infection,cancer, genetic disorder, or autoimmune disease) is observed and/ordetected upon or after administration of a substance of the presentdisclosure to a subject. Treatment can range from a positive change in asymptom or symptoms of the disease to complete amelioration of thedisease (e.g., viral infection, bacterial infection, parasiticinfection, or cancer) (e.g., reduction in severity, intensity, orduration of disease, alteration of clinical parameters indicative of thesubject's condition, relief of discomfort or increased or enhancedfunction), as detected by art-known techniques. The methods of thepresent disclosure can be utilized, for instance, to prevent or treat aviral infection, bacterial infection, parasitic infection, or cancer.One of skill in the art would recognize that this viral infection,bacterial infection, parasitic infection, or cancer can includeconditions characterized by the presence of a foreign pathogen orabnormal cell growth. Clinical symptoms will depend on the particularcondition and are easily recognizable by those skilled in the art oftreating the specific condition. Treatment methods can include, but arenot limited to therapeutic vaccinations. Thus, disclosed are methods oftreating a subject with a condition comprising administering to thevector or other composition disclosed herein.

Also disclosed are methods wherein the condition being treated orprevented is a viral infection. The viral infection can be selected fromthe list of viruses consisting of Herpes simplex virus type-1, Herpessimplex virus type-2, Cytomegalovirus, Epstein-Barr virus,Varicella-zoster virus, Human herpesvirus 6, Human herpesvirus 7, Humanherpesvirus 8, Variola virus, Vesicular stomatitis virus, Hepatitis Avirus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus,Hepatitis E virus, Rhinovirus, SARS, Coronavirus, Influenza virus A,Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus,Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus,Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fevervirus, Ebola virus, Marburg virus, Lassa fever virus, Eastern EquineEncephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitisvirus, Murray Valley fever virus, West Nile virus, Rift Valley fevervirus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, SimianImmunodeficiency virus, Human T-lymphotropic virus type-1, HumanT-lymphotropic virus type-2, Primate T-lymphotropic virus, Hantavirus,Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiencyvirus type-1, and Human Immunodeficiency virus type-2.

Also disclosed are methods wherein the condition being treated orprevented is a bacterial infection. The bacterial infection can beselected from the list of bacterium consisting of M. tuberculosis, M.bovis, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare,M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspeciesparatuberculosis, Nocardia asteroides, other Nocardia species,Legionella pneumophila, other Legionella species, Salmonella typhi,other Salmonella species, Shigella species, Yersinia pestis, Pasteurellahaemolytica, Pasteurella multocida, other Pasteurella species,Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeriaivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium,Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci,Coxiella burnetti, other Rickettsial species, Ehrlichia species,Staphylococcus aureus, Staphylococcus epidermidis, Streptococcuspyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichiacoli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis,Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species,Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species,Clostridium tetani, other Clostridium species, Yersinia enterolitica,and other Yersinia species.

Also disclosed are methods wherein the antigen-encoding nucleic acid isan antigen from a bacterium. The bacterial antigen can be selected fromthe group consisting of M. tuberculosis, M. bovis, M. bovis strain BCG,BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii,M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardiaasteroides, other Nocardia species, Legionella pneumophila, otherLegionella species, Salmonella typhi, other Salmonella species, Shigellaspecies, Yersinia pestis, Pasteurella haemolytica, Pasteurellamultocida, other Pasteurella species, Actinobacillus pleuropneumoniae,Listeria monocytogenes, Listeria ivanovii, Brucella abortus, otherBrucella species, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydiatrachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsialspecies, Ehrlichia species, Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillusanthracis, Escherichia coli, Vibrio cholerae, Campylobacter species,Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi,other Hemophilus species, Clostridium tetani, other Clostridium species,Yersinia enterolitica, and other Yersinia species.

Also disclosed are methods wherein the condition being treated orprevented is a fungal infection. The fungal infection can be selectedfrom the list of fungus consisting of Candida albicans, Cryptococcusneoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodesimmitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis,Pneumocystis carinii, Penicillium marneffi, and Alternaria alternatas.

Also disclosed are methods wherein the condition being treated is aparasitic infection. The parasitic infection can be selected from thelist of parasites consisting of Toxoplasma gondii, Plasmodiumfalciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodiumspecies, Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, otherLeishmania species, Schistosoma mansoni, other Schistosoma species, andEntamoeba histolytica.

In addition, the disclosed vectors and vector containing compositionscan be used to treat any disease where uncontrolled cellularproliferation occurs, such as a cancer. A non-limiting list of differenttypes of cancers that can be treated with the disclosed compositions isas follows: lymphomas (including Hodgkins and non-Hodgkins, B celllymphoma, and T cell lymphoma), mycosis fungoides, leukemias (includingmyeloid leukemia), carcinomas, carcinomas of solid tissues, squamouscell carcinomas, adenocarcinomas, sarcomas, gliomas, high grade gliomas,bladder cancer, brain cancer, nervous system cancer, head and neckcancer, blastomas, neuroblastomas, plasmacytomas, histiocytomas,melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas orsarcomas, kidney cancer, lung cancers such as small cell lung cancer andnon-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer,pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma,squamous cell carcinomas of the mouth, throat, larynx, and lung, largebowel cancer, hematopoietic cancers; testicular cancer; colon and rectalcancers, prostatic cancer, or pancreatic cancer, cervical cancer,cervical carcinoma, breast cancer, and epithelial cancer, renal cancer,genitourinary cancer, pulmonary cancer, esophageal carcinoma, metastaticcancers, or cancers in general.

Also disclosed are methods wherein the antigen-encoding nucleic acid isa tumor antigen. The tumor antigen can be selected from the listconsisting of human epithelial cell mucin (Muc-1; a 20 amino acid corerepeat for Muc-1 glycoprotein, present on breast cancer cells andpancreatic cancer cells), the Ha-ras oncogene product, p53,carcino-embryonic antigen (CEA), the raf oncogene product, gp100/pmel17,GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, gp75,Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7,prostate-specific antigen (PSA), HPV-16, MUM, alpha-fetoprotein (AFP),CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7, Wilm'stumor antigen-1, telomerase, and melanoma gangliosides.

Disclosed are methods of treating a condition in a subject comprisingadministering to the subject the vector of the disclosure, wherein thecondition is due to a mutated, disregulated, disrupted, or deleted gene;autoimmunity; or inflammatory diseases, including but not limited tocystic fibrosis, asthma, multiple sclerosis, muscular dystrophy,diabetes, tay-sachs, spinobifida, cerebral palsy, parkinson's disease,Lou Gehrig's disease, alzheimer's, systemic lupus erythamatosis,hemophelia, Addsion's disease, Cushing's disease.

By “preventing” is meant that after administration of a substance of thepresent disclosure to a subject, the subject does not develop thesymptoms of the viral, bacterial, or parasitic infection, and/or doesnot develop the viral, bacterial, or parasitic infection. “Preventing”or “prevention” can also refer to the ultimate reduction of aninfection, condition, or symptoms of an infection, or condition relativeto infections or conditions in subjects that do not receive thesubstance. Methods of prevention can include, but are not limited toprofilactic vaccination. As such, disclosed are methods of preventing aninfection in a subject comprising administering to the subject thevector of the disclosure.

Also disclosed are methods of the disclosure, wherein the infectionprevented is a fungal infection or the antigen-encoding nucleic acid isan antigen from a fungus. The fungal infection or antigen can beselected from the list of Candida albicans, Cryptococcus neoformans,Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis,Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystiscarinii, Penicillium marneffi, and Alternaria alternata.

Also disclosed are methods of the disclosure, wherein theantigen-encoding nucleic acid is an antigen from a parasite. Theparasitic antigen can be selected from the group consisting ofToxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodiummalariae, other Plasmodium species, Trypanosoma brucei, Trypanosomacruzi, Leishmania major, other Leishmania species, Schistosoma mansoni,other Schistosoma species, and Entamoeba histolytica.

Also disclosed are methods of the disclosure, wherein the subject is ahorse, cow, pig, dog, car, mouse, monkey, human, or a cell isolated fromsuch an animal.

XXIII. Screening Methods

Disclosed herein are methods of identifying new primate T-lymphotropicviruses comprising: a) contacting a nucleic acid using a first set ofprimers and a second set of primers internal to the first set ofprimers, wherein the first set of primers is SEQ ID NOs: 19 and 20, andwherein the second set of primers is SEQ ID NOs: 21 and 22 underconditions that permit primer extension; b) identifying any amplifiednucleic acid; and c) comparing the sequence to known primateT-lymphotropic viral sequences, wherein a sequence divergence greaterthan 5% indicates a new virus.

Also disclosed are methods of identifying new primate T-lymphotropicviruses comprising: a) contacting a nucleic acid using a first set ofprimers and a second set of primers internal to the first set ofprimers, wherein the first set of primers is selected from the group ofprimers pairs consisting of SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 12,SEQ ID NOs: 15 and 16, SEQ ID NOs: 23 and 24, SEQ ID NOs: 27 and 28, SEQID NOs: 31 and 32, SEQ ID NOs: 69 and 70, SEQ ID NOs: 73 and 74, and SEQID NOs: 77 and 78, wherein the second set of primers is selected fromthe group consisting of SEQ ID NOs: 9 and 10, SEQ ID NOs: 13 and 14, SEQID NOs: 17 and 18, SEQ ID NOs: 25 and 26, SEQ ID NOs: 29 and 30, SEQ IDNOs: 33 and 34, SEQ ID NOs: 71 and 72, SEQ ID NOs: 75 and 76, and SEQ IDNOs: 79 and 80; b) identifying any amplified nucleic acid; and c)comparing the sequence to known primate T-lymphotropic viral sequences,wherein sequence divergence greater than 5% indicates a new virus.

It is also understood that the disclosed methods of identifying a newprimate T-lymphotrophic virus can be achieved using non-nested PCRtechniques such as real-time PCR. Thus, for example, specificallydisclosed are methods of identifying new primate T-lymphotropic virusescomprising a) contacting a nucleic acid using a set of primers, whereinthe set of primers is selected from the set of primers consisting of SEQID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 7 and 8, SEQ IDNOs: 11 and 12, SEQ ID NOs: 15 and 16, SEQ ID NOs: 23 and 24, SEQ IDNOs: 27 and 28, SEQ ID NOs: 31 and 32, SEQ ID NOs: 69 and 70, SEQ IDNOs: 73 and 74, SEQ ID NOs: 77 and 78, SEQ ID NOs: 9 and 10, SEQ ID NOs:13 and 14, SEQ ID NOs: 17 and 18, SEQ ID NOs: 25 and 26, SEQ ID NOs: 29and 30, SEQ ID NOs: 33 and 34, SEQ ID NOs: 71 and 72, SEQ ID NOs: 75 and76, SEQ ID NOs: 79 and 80, and SEQ ID NOs: 64 and 65; b) identifying anyamplified nucleic acid; and c) comparing the sequence to known primateT-lymphotropic viral sequences, wherein sequence divergence greater than5% indicates a new virus. Also disclosed are identification methodswherein the method is a real-time PCR method.

Furthermore, the disclosed methods can be used in conjunction withprobes to detect the presence of amplification product. Specificallydisclosed are fluorescently labeled probes that can be used to detectthe amplification product of the disclosed methods. For example, afluorescent probe, can comprise TTCCCCAAGGCTTCAAAAACAGCCCCACGC (SEQ IDNO: 66).

The surface antigen (SU) and transmembrane regions of env can be usedserologically for the identification and differentiation of PTLVs (thetype specific peptides MTA-1 and K55 are in SU; likewise the p24 regionof gag can be used for the serological identification of PTLV). Thus,disclosed herein are methods of identifying a PTLV comprising contactinga nucleic acid with a set of primers specific for the surface antigen ortransmembrane regions of env and identifying any amplified nucleic acid.

In addition, the disclosed peptides, polypeptides, proteins and proteinfragments can be used to generate antibodies that can be used toidentify new and known primate T-lymphotropic viruses. Specificallydisclosed are methods of identifying the presence of a primateT-lymphotropic virus in a subject comprising taking a tissue sample fromthe subject and contacting the sample with an antibody directed to anHTLV-3 or HTLV-4 peptide, polypeptide, protein, or protein fragment,wherein the peptide, polypeptide, protein, or protein fragment can beSEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 46, 48, 50, 54, 55, 56, 57,58, 60, 62, 67, or 68, or the polypeptide, protein, or protein fragmentencoded by the nucleic acid of SEQ ID NO: 1, 2, 3, 4, 5, 6, 35, 36, 45,47, 49, 51, 52, 53, 59, 61, 63, or 81, and wherein binding of theantibody to the sample indicates the presence of a new or known primateT-lymphotropic virus. The disclosed methods also can be used to identifynew primate T-lymphotropic viruses as well as detect all primateT-lymphotropic viruses or a group of particular primate T-lymphotropicviruses. Those of skill in the art will know which antibodies to use toaccomplish their detection goal. For example, to detect more than one ofthe known HTLV viruses (HTLV-1, 2, and 3, or HTLV-1, 2, and 4) one canuse type specific peptide of HTLV-1 and HTLV-2 such as SEQ ID NO: 67 and68.

Also provided is a method of screening a substance for effectiveness intreating or reducing the severity of the condition (e.g., HTLV-3 orHTLV-4 infection) comprising: a) obtaining an animal having thecondition or characteristic (e.g., symptom) of the condition; b)administering the substance to an animal having one or morecharacteristics of the condition; and assaying the animal for an effecton the condition, thereby identifying a substance effective in reducingthe condition. The ability of a substance to reduce the severity of acondition can be determined by evaluating the histological and/orclinical manifestations of the condition before and after administrationof the substance of interest, and quantitating the degree of reductionof the histological and/or clinical manifestations of the condition. Theanimal in which the condition or characteristic (e.g., symptom) of thecondition is produced can be any mammal, and can include but is notlimited to mouse, rat, guinea pig, hamster, rabbit, cat, dog, goat,monkey, and chimpanzee. The condition or characteristic (e.g., symptom)of the condition can be produced in the animal by any method known inthe art. For example, HTLV-3 or HTLV-4 can be produced by introducinginto the animal (e.g., a chimpanzee infected with HTLV-3 or HTLV-4 orrhesus macaques or nemestrina macaques infected with an HTLV-3 or HTLV-4env on an SIV backbone. Pullium et. al., J. Infectious Dis. 183:1023,2001) an infectious amount of HTLV-3 or HTLV-4.

The present disclosure also provides a method of screening for asubstance effective in preventing the condition (e.g., HTLV-3 or HTLV-4infection) comprising: a) administering the substance to an animalsusceptible to the condition; b) subjecting the animal to treatment thatwill induce the condition or characteristic (e.g., symptom) of thecondition; and c) assaying cells from the animal for an change in immuneresponses as compared to an the immune responses in a control animalhaving the condition in the absence of the substance identifies asubstance that is effective in preventing the condition.

Also provided is a model for use in screening for substances effectivein treating or preventing a disease comprising an animal capable ofmanifesting a characteristic of the disease, wherein the animal has beenadministered the vector of the disclosure.

Further embodiments are methods of making a model of HTLV-3 or HTLV-4infection, comprising obtaining an animal capable of manifesting acharacteristic of the disease, and administering to said animal one ofthe vectors disclosed herein that encodes an antigen associate with thedisease. Also disclosed is a method of screening for a substanceeffective in treating a disease associated with an immunizing construct,the method comprising: a) administering the substance to the model ofthe disclosure; and b) assaying for an change in the course of thedisease as compared to an the course of the disease in a controlsubject. An improvement in the course of the disease in the presence ofthe substance identifies a substance that is effective in treating thedisease.

Still other embodiments are methods of screening for a substanceeffective in preventing a disease associated with an immunizingconstruct, the method comprising: a) administering one of the vectorsdisclosed herein to a subject; b) subjecting the subject to treatmentthat will induce the disease or characteristic (e.g., symptom) of thedisease; and c) assaying for an change in the course of the disease ascompared to an the course of the disease in a control subject. Animprovement in the course of the disease in the presence of thesubstance identifies a substance that is effective in preventing thedisease.

Yet still other embodiments are methods of screening for a substanceeffective in treating a disease associated with an immunizing construct,the methods comprising: a) subjecting a subject to treatment thatinduces the disease or characteristic (e.g., symptom) of the disease; b)administering to the subject one of the vectors disclosed herein; and c)assaying for a change in the course of the disease as compared to an thecourse of the disease in a control subject. An improvement in the courseof the disease in the presence of the substance identifies a substancethat is effective in treating the disease.

XXIV. Methods of Using the Disclosed Compositions as Research Tools

The disclosed compositions can also be used diagnostic tools related toprimate T-lymphotropic diseases such as HTLV-3 and HTLV-4.

XXV. Methods of Making the Compositions

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

XXVI. Processes for Making the Compositions

Disclosed are processes for making the disclosed compositions as well asmaking the intermediates leading to the compositions. For example,disclosed are nucleic acids in SEQ ID NOs: 1-6, 35, 36, 45, 53, and 81.There are a variety of methods that can be used for making thesecompositions, such as synthetic chemical methods and standard molecularbiology methods.

In some embodiments, a nucleic acid molecule is produced by the processof linking in an operative way a nucleic acid comprising the sequenceset forth in SEQ ID NOs: 1-6, 35, and 45 and a sequence controlling theexpression of the nucleic acid. In certain examples, the nucleic acidmolecule is produced by linking in an operative way a nucleic acidmolecule comprising a sequence having 80% identity to a sequence setforth in SEQ ID NOs: 1-6, 35, and 45, and a sequence controlling theexpression of the nucleic acid. In other embodiments, the nucleic acidmolecule is produced by linking in an operative way a nucleic acidmolecule comprising a sequence that hybridizes under stringenthybridization conditions to a sequence set forth SEQ ID NOs: 1-6, 35,and 45 and a sequence controlling the expression of the nucleic acid.

In other embodiments, a transformed cell is produced by transforming thecell with any of the nucleic acids disclosed herein, for example, any ofthe non-naturally occurring nucleic acids disclosed herein. In stillother embodiments the peptides disclosed herein are produced byexpressing any of the disclosed nucleic acids.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1

To determine whether HTLVs are present among individuals exposed to theblood and body fluids of wild primate populations (Wolfe et al. 2004a)known to be infected with STLV (Courgnaud et al. 2004), individuals wereexamined from twelve villages in southern Cameroon proximal to bothforested and nonforested NHP habitats. Individuals were asked toidentify and quantify their exposure to NHPs, which were organizedaccording to three categories reliably distinguished by this population:chimpanzee, gorilla and monkey (Wolfe et al. 2004a). A total of 930 whoreported exposure to NHP blood and body fluids, mainly through huntingand butchering were selected for further analysis. Plasma specimens fromexposed people were screened for PTLVs using an HTLV-1/2 EIA (ELISAImmunoassay) capable of detecting antibodies to a broad range of PTLVs,followed by confirmation with an HTLV Western blot (WB) assay that candistinguish HTLV-1 and HTLV-2 (van Dooren et al. 2004). A total of 97(10.4%) persons were EIA reactive of which 90 (9.7%) were also reactivein the WB assay. A broad range of WB profiles were seen, includingHTLV-1-like (1.1%), HTLV-2-like (0.5%), HTLV-positive but untypeable(1.4%), and HTLV indeterminate (6.7%).

DNA from peripheral blood mononuclear cells (PBMCs) available from 86 ofthe 90 WB reactive samples were then subjected to PCR amplification ofseveral viral regions. Viral sequences from 13 persons were obtainedusing this strategy. The WB reactivities of these 13 persons is shown inFIG. 1 and included HTLV-1-like (n=9), HTLV-2-like (n=1), and HTLVindeterminate (n=3) profiles. All 13 HTLV-infected persons wereexclusively from lowland forest sites, including both men and women whooften reported multiple opportunities for contact with the blood andbody fluids of NHPs (Table 3). Since PTLV diversity is influenced moreby geography than by primate species (Salemi et al 1999, Slattery et al.1999, Gessain & Mahieux 2000), viral sequences were analyzedphylogenetically along with African and global representatives of HTLVand STLV.

TABLE 3 Nonhuman primate exposures for human T-lymphotropic virus(HTLV)-infected central African hunters NHP Exposure Hunting HuntButcher Pet Reported ID Site HTLV Nearest PTLV Sex Age Technique m c g mc g m c g Injuries 1842 LE HTLV-1 Group D - m 32 x Mandrill clade 1863LE HTLV-4 Distinct from all m 48 S x x x bitten/ major PTLV scratchedgroups by wild animal 2472 LE HTLV-1 Group A f 27 x Cosmopolitan 979 MOHTLV-1 Group B- Central m 30 G x x monkey African bite 1127 MO HTLV-1Group D - m 44 g, s x x x x Mandrill clade 1380 MV HTLV-1 Group B-Central f 55 x x x African 1443 MV HTLV-1 Group B- Central f 71 x x xAfrican 1503 MV HTLV-1 Group B- Central f 75 x x x African 1537 MVHTLV-1 Group B- Central m 39 g, s x x wild African animal injured finger2026 ND HTLV-3 STLV-3 m 63 S x x 2656 ND HTLV-1 Group G - Central m 65 Gx x x West Africa 1259 NG HTLV-1 Group D - m 71 g, s x x bitten/Mandrill clade scratched by wild animal 2810 YI HTLV-1 Group G - Centralm 55 S x x West Africa *, m = monkey, c = chimpanzee, g = gorilla. †,PTLV, primate T lymphotropic virus; STLV, simian T-lymphotropic virus

TABLE 4 Nucleotide and Amino Acid Percent Identities¹ HTLV-1 HTLV-2STLV-2 STLV-3 HTLV-3 (ATK) (MoT) (PP1664) (TGE2117) (2026ND)HTLV-3_(2026ND) Genome (8917-bp) 61.6 62.9 62.6 87.0 — LTR(697-bp) 48.743.7 41.4 86.7 — gag (1268-bp) 69.3(83.2) 69.4(80.5) 70.6(80.7)87.5(96.0) — pro (534-bp) 59.7(62.6) 59.2(66.7) 59.4(59.3) 84.3(88.1) —pol (2670-bp) 62.2(66.2) 63.9(71.2) 63.5(69.9) 86.2(93.1) — env(1476-bp) 65.9(73.8) 69.0(78.2) 67.1(77.4) 87.8(95.7) — tax (1053-bp)76.3(81.4) 75.1(83.4) 74.4(80.4) 91.2(97.4) — rex (549-bp) 76.9(61.9)76.3(60.6) 75.8(63.5) 87.6(89.6) — pX (699-bp) 43.3 50.5 49.8 85.6 —HTLV-4_(1863LE) Genome (5320-bp) 64.0 72.2 71.4 66.2 66.1 pro (273-bp)²71.4(55.6) 79.5(28.1) 79.5(36.0) 71.8(29.2) 73.3(31.7) pol (2549-bp)²63.6(68.7) 71.4(80.1) 71.0(79.7) 65.2(71.7) 64.8(71.6) env (1458-bp)65.8(75.9) 73.1(85.3) 72.0(85.5) 67.2(78.8) 68.5(79.4) tax (765-bp)²77.4(85.1) 81.7(92.6) 79.4(92.9) 75.2(86.7) 75.0(86.3) rex (512-bp)76.0(63.9) 79.5(74.1) 80.7(68.8) 72.5(57.7) 72.7(59.4) pX (559-bp) 46.160.8 59.9 53.6 51.3 ¹Amino acid identiities are in parentheses. ²Onlypartial sequences are available

Most notable of the findings was the discovery of a human virus that isdistinct from all known PTLV lineages with 26-34% and 18-25% nucleotidedivergence in the conserved pol and tax genes, respectively, a range ofnucleotide divergence similar to that seen between HTLV-1, HTLV-2, andSTLV-3 (Meertens et al. 2002; Table 4). This virus formed a separatephylogenetic lineage with a long branch length and significant bootstrapsupport in both the pol (FIG. 2 a: pol tree) and tax trees. Phylogeneticanalyses combined with GenBank blast searches show that this is the onlyknown virus in this novel group. For these reasons this virus, which isdesignated HTLV-4, qualifies as the first member of a novel species inthe deltaretrovirus genus. Following ICTV guidelines (van Regenmortel2000) and pending formal classification, it is proposed that primateT-lymphotropic virus 4 (PTLV-4) be the name for this species, withPTLV-4(1863LE) as the prototype strain. HTLV-4 was found in a 48 yearold male hunter (1863LE) from the southern forests of Cameroon who hadan HTLV-2-like WB result and reported hunting monkeys, chimpanzees, andgorillas, and also being bitten and scratched by a wild animal, althoughthe animal causing the injury was not specified.

Also documented, with significant phylogenetic bootstrap support, is thefirst evidence of human infection within the PTLV-3 group (FIG. 2 a: poltree). This virus, which is designated HTLV-3, clusters with STLV-3viruses present in West African NHPs as expected (FIG. 2 d: LTR tree).HTLV-3 was found in a 63 year old male (2026ND) from the southernforests of Cameroon who had an HTLV-1-like WB result and who reportedhunting and butchering of monkeys. The fact that this virus falls withinthe diversity of a group of STLVs first identified in 1994 (Goubau et al1994) without evidence of a human counterpart to date, indicates thatthis infection was most likely acquired zoonotically through exposure tothe blood or body fluids of a hunted NHP from this region (Courgnaud etal. 2004).

In addition, broad diversity of HTLV-1 viruses was also found in thiscollection. Of the 11 HTLV-1 sequences, two did not fall within any ofthe known HTLV-1 subtypes but clustered clearly within a clade thatincluded only STLV-1 from central and west Africa (FIG. 2 d: LTR tree).One of these viruses clustered with STLV-1 from monkeys in Cameroon andwas from a 65 year-old male (2656ND) from the southern forest zone ofCameroon. He reported hunting and butchering of monkeys and kept agorilla as a pet (Table 3). The second virus clustered with STLV-1recently identified in chimpanzees and red colobus monkeys (Leendertz etal. 2004) and was from a 55 year old male (2810YI) who reported huntingand butchering of monkeys (Table 3). The presence of these viruses inhunters, seen previously only among NHPs, indicates that these personswere infected zoonotically. This distinct clade is referred to as HTLV-1subtype G. Three subjects (1259NG, 1127MO, 1842LE) from differentvillages were found to have HTLV-1 subtype D, viruses known to infectgeographically overlapping populations of humans and mandrills incentral Africa (FIG. 3 LTR tree (Mahieux et al. 1998). Two of the threeviruses were found in hunters (Table 3), providing indirect evidence ofcross-species transmission between humans and mandrills within subtype Dand supporting further the claims of cross-species transmission of thissubtype (Mahieux et al. 1998). These results are consistent with SFVinfection from mandrills that was documented previously in thispopulation (Wolfe et al 2004) and indicate that the frequent hunting ofmandrills may explain the widespread transmission of mandrillretroviruses. Five persons (979MO, 1380MV, 1443MV, 1503MV, 1537MV) wereinfected with HTLV-1 subtype B viruses, which are known to be endemicamong humans in central Africa and which are believed to have originatedfrom STLV-1 in this region (Mahieux, R. et al 1997, Gessain, A. &Mahieux, R 2000; FIG. 3: LTR tree). Thus, these five new subtype Bviruses may have been acquired either zoonotically from STLV-1-infectedprimates or from human-to-human transmission, or both.

Notably, a 71 year old female (1443MV) who reported butchering gorillaswas found to be infected with a virus most closely related to STLV-1found in two gorillas from Cameroon (Nerrienet 2004, Courgnaud et al2004), although without significant bootstrap support (FIG. 3 LTR).Interestingly, person 1503MV is also WB positive for SFV (Wolfe et al2004), indicating that zoonotic transmission in an individual is notlimited to a single retrovirus and providing a biological setting forviral recombination and altered pathogenicity and transmissibility ofthese viruses. One person (2472LE) was infected with the HTLV-1 subtypeA virus, a clade consisting of sequences from only globally disseminatedHTLV-1 and thus this infection was most likely acquired throughhuman-to-human transmission. DNA samples from the remaining 73 personswith reactive WB results were all negative by the generic PCR assay fortax sequences and four other sequences specific for each PTLV clade,including HTLV-4. The results demonstrate that HTLV diversity is fargreater than previously understood. The data indicate that contact withthe blood and body fluids of NHPs is a major factor in the emergence ofnovel HTLVs, which are known to be transmissible among humans and havethe potential to cause disease. Because the hunting and butchering ofwild NHPs is widespread throughout central Africa (Bowen-Jones & Pendry1999) and STLVs are known to be highly prevalent among hunted NHPs(Courgnaud et al. 2004), it is suspected that zoonotic transmission ofSTLV is not a restricted risk. Since blood banks in central Africa donot generally screen for HTLV, further spread of these viruses amongcentral Africans may be facilitated by blood donations from infectedpersons. That HTLV-4 represents a previously unrecognized virus beingtransmitted between humans indicates that more substantial screening forthis virus in central African populations is needed. The finding thatboth HTLV-4 and HTLV-3 are serologically indistinguishable from HTLV-1and HTLV-2 in current assays can explain why these viruses have not beenpreviously identified, and highlight the importance of improveddiagnostic assays. The increasing evidence that primate hunting isassociated with the emergence of a range of simian retroviruses (Wolfeet al. 2004b) calls for increased surveillance and follow-up ofindividuals exposed to the blood and body fluids of wild NHPs, and foreffective strategies to control the hunting of NHPs.

Methods

Ethical Approvals

Studies were conducted in the context of a community-based HIVprevention campaign designed to provide information using Cameroonianeducators and counselors and therefore to decrease transmission.Participation in the study was completely voluntary. The study protocolwas approved by the Johns Hopkins Committee for Human Research, theCameroon National Ethical Review Board, and the HIV Tri-ServicesSecondary Review Board. Questionnaires and matching samples wereanonymized by removing all personal identifiers to provide an unlinkedstudy population.

Sample Preparation and Serology

Blood was collected from participants, transported to a centrallaboratory, processed into plasma and PBMC aliquots and stored at −80°C. Initial screening for HTLV antibodies in serum and plasma samples wasperformed by using the Vironostika HTLV-1/2 microelisa system(Organon-Teknika, Durham, N.C.) following the manufacturer'sinstructions. Reactive samples were then tested in a WB test (HTLV Blot2.4, Genelabs Diagnostics, Singapore) that contains disrupted HTLV-1virions, a gp21 recombinant protein (GD21) common to both HTLV-1 andHTLV-2, and two HTLV-type specific recombinant envelope (Env) peptides,MTA-1 and K55, which allow serological differentiation of HTLV-1 andHTLV-2, respectively. Samples with reactivity to the Gag (p24) and Env(GD21) proteins were considered seropositive. Seropositive samples withreactivity to MTA-1 or K55 were considered HTLV-1-like or HTLV-2-like,respectively. Samples with reactivity to either p24 or GD21 alone or incombination with other HTLV proteins (FIG. 1) were consideredindeterminate.

PCR and Sequence Analysis

DNA was prepared from uncultured PBMCs and its integrity was confirmedby β-actin PCR as previously described. All DNA preparation and PCRassays were performed in a laboratory where only human samples areprocessed and tested following recommended precautions to preventcontamination. DNA samples were first screened with a generic PTLV taxPCR assay capable of detecting 222-bp sequences from each of the threemajor PTLV groups (Busch et al. 2000, van Dooren et al. 2004). Sequenceanalysis of this tax sequence provided broad genetic classification intoeach PTLV group. Phylogenetic resolution within the PTLV-1 and PTLV-3groups was done using LTR sequences as described previously (van Doorenet al. 2004, Meertens et al. 2001). A portion of the 3′ HTLV-1 LTR fromselected samples (1259NG, 1127MO, 1842LE, and 2810YI) was amplified bynested PCR using external primers 5VLTRext 5′ AACCACCCATTTCCTCCCCATG 3′(SEQ ID NO: 19; Meertens et al. 2001) and 1MNDR15′GTCGTGAATGAAAGGGAAAGGGGT 3′ (SEQ ID NO: 20; Meertens et al. 2001), andthe internal primers Enh280 5′ TGACGACAACCCCTCACCTCAA 3′ (SEQ ID NO: 21;Meertens et al. 2001) and 1MNDR2 5′ AGGGGTGGAACTTTCGATCTGTAA 3′ (SEQ IDNO: 22; Meertens et al. 2001). The tax (577-bp) and polymerase (pol)(709-bp) sequences of HTLV-3 and HTLV-4 were amplified by nested PCRusing primers designed from conserved PTLV regions. The external andinternal tax primers are PTLVTPG 5′T(C/T)ACCT(G/A)GGACCCCATCGATGGACG 3′(SEQ ID NO: 7) and PGTAXR1 5′ GAIGA(T/C)TGI A(C/G)TAC(T/C)AAAGATGGCTG 3′(SEQ ID NO: 8) and PH2Rrev 5′ CCTTATCCCTCGICTCCCCTC CTT 3′ (SEQ ID NO:9) and PGTAXR2 5′ TTIGGG(T/C)AIGGICCGG AAATCAT 3′ (SEQ ID NO: 10),respectively. The external and internal pol primers are PGPOLF1 5′C(T/G)TTAAACCIGA(A/G)CGCCT CCAGGC 3′ (SEQ ID NO: 11) and PGPOLR1GG(T/C)(A/G)TGIA (A/G)CCA(A/G)(A/G)CIAG(T/G)GG CCA 3′ (SEQ ID NO: 12)and PGPOLF2 5′ AC(T/C)TGGT(C/T)(C/T) (G/C)(G/C)A(A/G)GGCCCTGGAGG 3′ (SEQID NO: 13) and PGPOLR2 5′ G(A/G)(T/C)(A/G)GGIGTIC CTTTIGAGACCCA 3′ (SEQID NO: 14), respectively. Inosines (I) and wobble bases (N/N) were usedto accommodate areas of heterogeneity (Table 5).

Additional diagnostic PCR with PTLV-specific primers was carried out onsamples with negative results for the generic 222-bp tax fragments.Assays described previously were used for PTLV-1 env and STLV-3 LTR (vanDooren et al. 2004) and HTLV-2 env (Switzer et al. 1995). For HTLV-4, anew nested PCR assay was developed based on the HTLV-4 tax sequenceusing the external primers 1863TF1 5′ CTCCTTCTTTCAGTCCGTGCGGAG 3′ (SEQID NO: 15) and 1863TR1 5′ GGGGTAGTCAGGTTTGGCTGGTAT 3′ (SEQ ID NO: 16)and the internal primers 1863TF2 5′ CCTACCGCAACGGATGTCTTGAAA 3′ (SEQ IDNO: 17) and 1863TR2 5′ TATGGCGCC GGTGTGATGATAAAG 3′ (SEQ ID NO: 18) andstandard conditions to generate a 275-bp fragment. Percent nucleotidedivergence was calculated using the Gap program in the Genetic ComputerGroup's Wisconsin package. Sequences were aligned using the Clustal Wprogram, gaps were removed, and distance-based trees were generated byusing the Kimura two-parameter model in conjunction with the NJ methodin the MEGA program (version 2.1) as described elsewhere (van Dooren etal 2004). 1000 bootstrap replicates were used to test the reliability ofthe final topology of the trees.

Primate Taxonomic Nomenclature

Nomenclature used herein was as described. NHPs were coded using thefirst letter of the genus and the first two letters of the species nameswith their house names or codes within parentheses. Cmo=Cercopithecusmona (Mona monkey), Cne=C. neglectus (De Brazza's guenon), Cmi=C. mitis(Sykes's monkey), Cni=C. nictitans (greater spot-nosed guenon),Cae=Chlorocebus species (African green monkey), Cpo=C. pogonias (crownedmonkey), Cto=Cercocebus torquatus (red-capped mangabey), Cag=Cercocebusagilis (agile mangabey), Mog=Miopithecus ogouensis (talapoin monkey),Ani=Allenopithecus nigrpyridis (Allen's swamp monkey), Msp=Mandrillussphinx (mandrill (mnd)), Pan=Papio anubis (olive baboon (bab)), Pcy=P.cynocephalus (yellow baboon), Pha=P. hamadryas (sacred baboon), Ppu=P.ursinus (chacma baboon), Ppa=P. papio (Guinea baboon), Pba=Piliocolobusbadius (red colobus monkey), Mto=Macaca tonkeana (Celebes macaque),Ptr=Pan troglodytes (chimpanzee), Ppn=Pan paniscus (bonobo), Ggo=Gorillagorilla (western lowland gorilla).

TABLE 5 Sequences of primers used for amplifying partial tax, envelope(env), polymerase (pol) and LTR regions of primate T-cell lymphotropicviruses Expected PCR Annealing Primer sequence^(a) product temp (°C.),Name (5′to 3′) SEQ ID NO. Location^(b) size (bp) No. of cycles PH1FTTGTCATCAGCCCACT (SEQ ID NO: 23) tax, 7243-7262, outer TCCCAGG PH2RAAGGAGGGGAGTCGAG (SEQ ID NO: 24) tax, 7478-7455, outer 236 50, 40GGATAAGG PH2F CCCAGGTTTCGGGCAA (SEQ ID NO: 25) tax, 7257-7280, innerAGCCTTCT PH2R^(c) AAGGAGGGGAGTCGAG (SEQ ID NO: 26) tax, 7478-7455, inner222 50, 40 GGATAAGG PTLVTPG T(C/T)ACCT(G/A)G (SEQ ID NO: 7) tax,7480-7504, outer GACCCCATCGATGG ACG PGTAXR1 GAIGA(T/C)TGIA (SEQ ID NO:8) tax, 8140-8115, outer 660 45, 40 (C/G)TAC(T/C)A AAGATGGCTG PH2RrevCCTTATCCCTCGICTC (SEQ ID NO: 9) tax, 7529-7552, inner CCCTCCTT PGTAXR2TTIGGG(T/C)AIGGI (SEQ ID NO: 10) tax, 8106-8085, inner 577 45, 40CCGGAAATCAT PGPOLF1 C(T/G)TTAAACCIGA (SEQ ID NO: 11) pol,2611-2634,outer (A/G)CGCCTCCAGGC PGPOLR1 GG(T/C)(A/G)TGIA (SEQ ID NO: 12) pol,3598-3575, outer 987 45, 40 (A/G)CCA(A/G) (A/G)CIAG(T/G)GG CCA PGPOLF2AC(T/C)TGGT(C/T) (SEQ ID NO: 13) pol, 2643-2666, inner (C/T)(G/C)(G/C)A(A/G)GGCCCTGGAGG PGPOLR2 G(A/G)(T/C)(A/G) (SEQ ID NO: 14) pol,3352-3329, inner 709 45, 40 GGIGTICCTTTIGAGA CCCA PGENVF1 TGGATCCCGTGG(SEQ ID NO: 27) env, 5114-5136, outer (A/C)GI(C/T)TCCT IAA PGENVR1GT(A/G)TAIG(C/G) (SEQ ID NO: 28) env, 5576-5552, outer 462 45, 40(A/G)(C/G)AIGTCC AIG(A/C)(T/C)TGG PGENVF2 AIAGACC(T/A) (SEQ ID NO: 29)env, 5186-5209, inner (C/T)CAAC(A/T)CC ATGGGTAA PGENVR2 G(A/C)(T/C)TGGCA(SEQ ID NO: 30) env, 5557-5535, inner 371 45, 40 ICCIA(A/G)GTAIGG GCAGPLTRF1 (G/A)CCACCAICTIG (SEQ ID NO: 31) LTR, 8256-8282, outerIGGACAAATAGCTGA GPLTRR2 C(C/T)GGGCCAAGCC (SEQ ID NO: 32) LTR, 8830-8807,outer 575 45, 40 TCGCTGCAGGCA GPLTRF2 ACCIIGGCTCTGACGT (SEQ ID NO: 33)LTR, 8333-8356, inner CTCTCCCT GPLTRR2 GGCAGIAGAAGTGCTA (SEQ ID NO: 34)LTR, 8810-8787, inner 478 45, 40 CTTTCGAT ^(a)Inosines and wobblenucleotides were included in the primers to accommodate sequenceheterogeneity. ^(b)The positions of the the pol, env, and tax primersare given according to human T-cell lymphotropic virus type 1 (strainATK); the LTR primer positions are given according to the simian T-celllymphotropic virus type 3 (strain PH969) genome. ^(c)The primer PH2R isused with PH2F in a semi-nested PCR.Nucleotide Sequence Accession Numbers

The GenBank accession numbers for the 28 new HTLV sequences includeAY818406 and AY818433.

Example 2 Ancient Origin and Molecular Features of the HumanT-Lymphotropic Virus Type 3 Revealed by Complete Genome Analysis

Comparison of the HTLV-3(2026ND) Proviral Genome with Prototypical PTLVs

Using a combination of primers designed from small sequences obtained ineach of the three major genes of PTLV and the LTR region, the completegenome of HTLV-3(2026ND) was successfully generated as depicted in FIG.4. Sequence analysis of the overlapping regions, followed by comparisonwith the genetic structure of other PTLVs, demonstrated that thecomplete proviral genome of HTLV-3(2026ND) is 8917-bp. Despite beinggenetically equidistant from HTLV-1 and HTLV-2, the genomic structure ofHTLV-3(2026ND) was similar to that of other PTLVs and included thestructural, enzymatic, and regulatory proteins all flanked by longterminal repeats (LTRs). Comparison of HTLV-3(2026ND) with prototypicalPTLV genomes demonstrates that this new human virus is equidistant fromthe PTLV-1 (62% identity) and PTLV-2 (63% identity) groups across thegenome. The results also confirm that HTLV-3 has the closet nucleotideand protein sequence identity to STLV-3 (87-92% identity; Table 6).

TABLE 6 Percent Nucleotide and Amino Acid Identity of HTLV-3(2026ND)with other PTLV Prototypes¹ HTLV-1 HTLV-2 STLV-2 STLV-3 STLV-3 STLV-3STLV-3 (ATK) (MoT) (PP1664) (PH969) (PPAF3) (CTO604) (NG409) Genome 61.662.9 62.6 86.7 92.0 88.4 90.6 LTR 48.7 43.7 41.4 86.2 91.1 86.9 86.9 gag69.3 (83.2) 69.4 (80.5) 70.6 (80.7) 86.4 (95.5) 91.3 (97.6) 89.4 (96.2)90.6 (96.7) p19 (74.4) (68.3) (67.2) (95.9) (95.9) (95.9) (94.3) p24(90.1) (90.1) (90.6) (98.1) (99.1) (98.6) (99.1) p15 (78.0) (73.8)(72.6) (88.4) (96.5) (90.7) (94.2) pro 59.7 (62.6) 59.2 (66.7) 59.4(59.3) 83.3 (87.0) 88.8 (91.5) 85.0 (89.3) 88.0 (90.4) pol 62.2 (66.2)63.9 (71.2) 63.5 (69.9) 86.1 (92.7) 92.6 (94.9) 88.4 (92.9) 92.0 (92.9)env 65.9 (73.8) 69.0 (78.2) 67.1 (77.4) 88.1 (95.1) 92.3 (95.1) 88.4(94.3) 91.2 (95.3) SU² (68.4) (70.7) (69.7) (92.7) (97.1) (92.4) (94.0)TM² (83.5) (91.6) (91.0) (99.4) (98.9) (97.8) (97.8) rex 76.9 (61.9)76.3 (60.6) 75.8 (63.5) 87.1 (88.5) 90.9 (94.5) 88.5 (94.0) 88.3 (92.3)tax 75.4 (81.4) 73.1 (83.4) 72.3 (80.4) 90.2 (97.4) 94.0 (98.3) 91.4(96.6) 92.8 (96.9) ¹amino acid identity in parentheses; strain namesgiven in parentheses below PTLV designation ²SU, surface protein; TM,transmembrane protein

The most genetic divergence between the PTLV groups was seen in the LTRregion (52-59%) while the highest inter-group identity was observed inthe highly conserved regulatory genes, tax and rex (72-77%).Interestingly, within the PTLV-3 group, HTLV-3(2026ND), which wasidentified in a hunter from Cameroon, was unique but shared the mostoverall sequence identity to STLV-3(PPAF3) (92%) from a Senegalesebaboon instead of STLV-3(CT0604) (88.4%) identified in red-cappedmangabeys, also from Cameroon. This relationship is highlighted furtherby comparison of HTLV-3(2026ND) with all available full-length STLV-3genomes in similarity plot analysis where the highest identity was seenin the highly conserved tax gene. As seen within other PTLV groups,there was no clear evidence of genetic recombination of HTLV-3(2026ND)with STLV-3 or PTLV-1 and PTLV-2 proviral sequences by usingbootscanning analysis. HTLV-3(2026ND) was not compared to the recentlyreported second strain of HTLV-3 because only two short sequences wereavailable at GenBank and in these region this virus has been shown to benearly identical to STLV-3(CT0604) (Callatini et al. (2005)Retrovirology. 2:30).

Organization of the LTR and Pre-Gag Region

As with STLV-3, the HTLV-3(2026ND) LTR (697-bp) was smaller than that ofHTLV-1 (756-bp) and HTLV-2 (764-bp), by having two and not three of the21-bp transcription regulatory repeat sequences in the U3 region (FIG. 5a; Meertens and Gessain. (2003) J. Virol. 77:782-789; Meertens et al.(2002) J. Virol. 76:259-268; Van Brussel et al. (1997) J. Virol.7:5464-5472; Van Dooren et al. (2004) J. Gen. Virol. 85:507-519). Otherregulatory motifs such as the polyadenylation signal, TATA box, and capsite were all conserved in the HTLV-3(2026ND) LTR (FIG. 5 a). Bysecondary structure analysis of the LTR RNA sequence, a stable stem loopstructure from nucleotides 421-464 (FIG. 5 b) was also observed similarto that shown to be essential for Rex-responsiveness control of viralexpression in both HTLV-1 and HTLV-2.

Analysis of the Genomic Structure of HTLV-3(2026ND)

Translation of predicted protein open reading frames (ORFs) across theviral genome identified all major Gag, Pol, Pro (protease), and Envproteins, as well as the regulatory proteins, Tax and Rex. Translationof the overlapping gag and pro and pro and pol ORFs occurs by one ormore successive-1 ribosomal frameshifts that align the different ORFs.The conserved slippage nucleotide sequence 6(A)-8 nt-6(G)-11nt-6(C) ispresent in the Gag-Pro overlap, while a point mutation in the Pro-Poloverlap slippage sequence (GTTAAAC (SEQ ID NO: 82) compared to TTTAAAC(SEQ ID NO: 83) in HTLV-1 and HTLV-2) was observed in HTLV-3(2026ND) butthe asparagine codon (AAC) crucial for the slippage mechanism wasunaffected.

The structural and group specific precursor Gag protein consisted of 422amino acids (aa) that is predicted to be cleaved into the three coreproteins p19 (matrix), p24 (capsid), and p15 (nucleocapsid) similar toHTLV-1, HTLV-2, and STLV-3. Across PTLVs, Gag was one of the mostconserved proteins with identities ranging from 81% and 83% for HTLV-1and PTLV-2, to 95% for STLV-3 supporting the observed cross-reactivityseen with PTLV-3 antisera in Western blot assays using HTLV-1 antigens.Within Gag, the capsid protein showed greater than 90% identity toHTLV-1, while the matrix and nucleocapsid proteins were more divergentsharing less than 78% identity to PTLV-1 and PTLV-2 indicating theirpotential use in serologic assays for discriminating the three majorPTLV groups.

The predicted size of the Env polyprotein is 491 aa, which is slightlyshorter than that found in STLV-3s (313 aa versus 314 and 315 forSTLV-3(PH969) and STLV-3(CTO-604) due to sequence variation at thecarboxy terminus of the surface (SU) protein. In contrast, thetransmembrane (TM) protein (178 aa) was highly conserved across allPTLVs supporting further the use of the recombinant HTLV-1 GD21 proteinspiked onto WB strips for the identification of divergent PTLVs. Despitethe weak reactivity of anti-HTLV-3(2026ND) antibodies to the HTLV-1 typespecific SU peptide (MTA-1; Wolfe et al. (2005) Proc. Natl. Acad. Sci.USA. 102:7994-7999) spiked onto WB strips, there was only 70.8% identityof MTA-1 to HTLV-3(2026ND), which is similar to the 68.8% identity ofMTA-1 to HTLV-2, demonstrating no clear correlation of WB profile andpredicted SU sequence.

The HTLV-1 and HTLV-2 Tax proteins (Tax1 and Tax2, respectively)transactivate initiation of viral replication from the promoter in the5′ LTR and are thus essential for viral expression (Feuer and Green.(2005) Oncogene. 24:5996-6004). Tax1 and Tax2 have also been shown to beimportant for T-cell immortalization, while the HTLV-3 Tax (Tax3) hasnot yet been characterized (Feuer and Green. (2005) Oncogene.24:5996-6004). Hence, the Tax3 sequences were compared with those ofprototypic HTLV-1, PTLV-2, and STLV-3s to determine if motifs associatedwith these functional characteristics are preserved. Alignment ofpredicted Tax3 sequences shows excellent conservation of the criticalfunctional regions, including the nuclear localization signal (NLS),cAMP response element (CREB) binding protein (CBP)/P300 binding motifs,and nuclear export signal (NES; HTLV-3 Tax is shown in FIG. 6). TheC-terminal transcriptional activating domain (CR2), essential forCBP/p300 binding, was also very conserved except for a single UV to Fmutation at position five of the motif compared to HTLV-1 and PTLV-2,respectively. However, this single amino acid change in the STLV-3 Taxhas recently been shown in transient transfection assays to have nodeleterious effect on viral transactivation (Chevalier et al. (2005)AIDS Res. Hum. Retrovir. 21:513 (Abs. P174)). Since the predicted CR2domain is conserved in Tax3, similar transactivation activity can beseen with HTLV-3.

Interestingly, although these important functional motifs are highlyconserved in PTLV, phenotypic differences of HTLV-1 and HTLV-2 Taxproteins have been observed leading to speculation that thesedifferences account for the different pathologies associated with bothHTLVs (Feuer and Green. (2005) Oncogene. 24:5996-6004). Recently, theC-terminus of Tax 1, and not Tax2, has been shown to contain a conservedPDZ domain present in cellular proteins involved in signal transductionand induction of the IL-2-independent growth required for T-celltransformation (Rousset et al. (1998) Oncogene. 6:643-654; Tsubata etal. (2005) Retrovirol. 2:46). The presence of a PDZ domain in PTLV-1 andits absence in PTLV-2 indicates a potential role of this motif in thephenotypic differences of the two viral groups. The consensus PDZ domainhas been defined as S/TXV-COOH, where the first amino acid is serine orthreonine, X is any amino acid, followed by valine and the carboxyterminus. Examination of the PTLV-3 Tax sequences showed that bothHTLV-3 and STLV-3 have predicted PDZ domains with the consensus sequenceS(P/S)V compared to T(E/D)V in PTLV-1 (the HTLV-3 PDZ domain is shown inFIG. 6).

Besides Tax and Rex, two additional ORFs coding for four proteins(p27^(I), p12^(I), p30^(II), and p13^(II) where I and II denote ORFI andORFII, respectively) have been identified in the pX region of HTLV-1(FIG. 4) and are important in viral infectivity and replication, T-cellactivation, and cellular gene expression (Bindhu et al. (2004) Front.Biosc. 9:2556-2576). Analysis of the pX region of HTLV-3(2026ND)revealed a total of four putative ORFs (named I-IV, respectively) codingfor 96, 122, 72, and 118 aa in length. While both ORFIII (72 aa) andORFIV (118 aa) shared identity to the ORFII of STLV-3 and HTLV-1 andSTLV-2/HTLV-2, respectively, and each contained two PXXP motifs, onlyORF III was leucine rich like that seen in the leucine zipper motifs ofORFI p12^(I) (Bindhu et al. (2004) Front. Biosc. 9:2556-2576). However,ORFIII did not share any sequence homology with p12^(I) and both ORFIand ORFII shared only weak sequence identity to miscellaneous cellularproteins available at GenBank. Interestingly, 22 of 28 (79%) amino acidsin ORFIV (pos 64-91) were identical among the ORFIIs of all PTLVsindicating a conserved functionality of this motif.

A protein termed the HTLV-1 basic leucine zipper ZIP (bZIP) factor (HBZ)was recently identified in translation of the complementary strand ofthe viral RNA genome between the env and tax/rex genes (Gaudray et al.(2002) J. Virol. 76:12813-12822). Although originally reported to beexclusive to PTLV-1 (Gaudray et al. (2002) J. Virol. 76:12813-12822),HBZ is conserved among PTLVs, including HTLV-3(2026ND) (HTLV-3 HBZ isshown in FIG. 7), demonstrating further the potential importance of thisprotein in viral replication and oncogenesis. The carboxy terminus ofthe HBZ ORF contains a 21 aa arginine rich region that is relativelyconserved in PTLV and known cellular bZIP transcription factors,followed by a leucine zipper region possessing five or four conservedleucine heptads in HTLV-1 and all other PTLVs, respectively. PTLV-1 has5 leucine heptads similar to that found in mammalian bZIP proteins,while PTLV-1 and PTLV-2 have four leucine heptads followed by leucineoctet. Of all PTLVs with full length genomes available at GenBank, onlyHTLV-2(MoT) did not have the full complement of leucine heptads but waslimited to the initial three leucine motifs due to a one nucleotidedeletion at position 6823 causing a frameshift in the predicted HBZsequence.

Phylogenetic Analysis

The genetic relationship of HTLV-3(2026ND) to PTLV-3 was confirmed byusing aligned full-length prototype sequences excluding the LTR region(FIG. 8 a). Phylogenetic analysis inferred three major PTLV groups withvery high bootstrap support (100%) with HTLV-1, HTLV-2 and HTLV-3 eachclustering in separate clades (FIG. 8 a). Within the PTLV-3 phylogroup,HTLV-3(2026ND) formed a separate lineage but clustered with highbootstrap support with STLV-3s from west central Africa (strains CTO604,CTO-NG409, and PPA-F3) indicating a possible primate origin for thishuman infection in this geographic region. The relationship of HTLV-3 toSTLV-3 was supported further by phylogenetic inference of identical treetopologies using an alignment of each major gene region (FIG. 8 b-8 d).The phylogenetic stability seen across the PTLV genome also demonstratesfurther the absence of major recombination events occurring in PTLVdespite evidence of dual infections in humans and primates (Courgnaud etal. (2004) J. Virol. 78 :4700-4709), compared to other retroviruses suchas HIV which undergo frequent recombination.

Dating the Origin of HTLV-3(2026ND) and Other PTLVs

The finding of HTLVs in three distinct clades indicates an ancient,independent evolution of these viruses. Hence, additional molecularanalyses was undertaken in order estimate the divergence times of thePTLV lineages. Although others have reported finding a clock-likebehavior of STLV-3 sequences (Meertens and Gessain. (2003) J. Virol.77:782-789; Meertens et al. (2002) J. Virol. 76:259-268; Meertens et al.(2003) J. Gen. Virol. 84:2723-2727), these results were not confirmedand instead found that PTLVs evolved at different rates by using analignment of full-length PTLV genomes sans LTR sequences. However,reliable retrovirus divergence times can be obtained by usingnonparametric rate smoothing of the sequences to relax the stringency ofa clock assumption followed by time calibration of the tree using avalue of 40,000-60,000 YA for the origin of the Melanesian HTLV-1(Sanderson (2003) Bioinformatics. 19:301-2; Switzer et al. (2005)Nature. 434:376-380; Van Dooren et al. (2004) J. Gen. Virol.85:507-519). By using these dates and methods, the mean evolutionaryrate for PTLV was estimated to be 1.12×10⁻⁶ (confidence interval6.82×10⁻⁷ to 1.56×10⁻⁶) substitutions/site/year, respectively, which isconsistent with rates determined previously both with and withoutenforcing a molecular clock (Lemey et al. (2005) Infect. Gen. Evol.5:291-298; Meertens and Gessain. (2003) J. Virol. 77:782-789; Meertenset al. (2002) J. Virol. 76:259-268; Meertens et al. (2003) J. Gen.Virol. 84:2723-2727; Salemi et al. (2000) Mol. Biol. Evol. 17:374-386;Van Dooren et al. (2004) J. Gen. Virol. 85:507-519). The meanevolutionary rate for HTLV-3(2026ND) is estimated to be 9.94×10⁻⁷(confidence interval 6.04×10⁻⁷ to 1.38×10⁻⁶). The PTLV ancestor wasestimated to have originated about 630,000-947,000 YA confirming anarchaic evolution of the primate deltaretroviruses (FIG. 9; Salemi etal. (2000) Mol. Biol. Evol. 17:374-386). The separation of PTLV-1 andPTLV-2 occurred about 579,077-867,458 YA, while HTLV-2 and STLV-2diverged around 191,621-286,730 YA (FIG. 9). The origin of all PTLV-3swas estimated to be between 63,294-94,700 YA with the ancestor ofHTLV-3(2026ND) occurring about 36,087-54,067 YA (FIG. 9) indicating anancient origin of this virus in humans. Alternatively, HTLV-3 mayrepresent a recent zoonoses from a primate infected with a very old,divergent STLV-3. However, if HTLV-3 is an old human infection, then itappeared during the same period as the ancestor of both HTLV-1 andHTLV-2 (40,000-60,000 and 28,800-43,392 YA, respectively) and may havealso spread to become endemic in specific populations yet to beidentified.

Discussion

The complete nucleotide sequence and genomic characterization of thefirst HTLV-3 that is clearly distinct from all STLV-3s and isgenetically equidistant to HTLV-1 and HTLV-2 is described herein.HTLV-3(2026ND) is also unique from the second HTLV-3(Py143) reportedrecently in a Bakola pygmy from Cameroon since the latter strain isnearly identical to STLV-3 found in a red-capped mangabey, based on thelimited sequence data available for this virus (Callatini et al. (2005)Retrovirology. 2:30). Although HTLV-1 and HTLV-2 are pathogenic and havespread globally to become endemic in different human populations, littleis known about the epidemiology of HTLV-3 infection. However, detailed,comparative sequence analyses of viral genomes can help provideimportant molecular clues to the origin, evolution, and public healthimportance of novel human infections.

Like other PTLVs, HTLV-3(2026ND) is genetically stable and its slowevolutionary rate, combined with estimates of known human migrations,can then be used to infer divergence times for HTLV. The finding thatthe predecessor of HTLV-3(2026ND) originated over 30 millenia ago, anage which is estimated that the ancestors of both HTLV-1 and -2 to haveappeared, combined with the wide geographic distribution of STLVs andthe recent finding of another HTLV-3 in an African pygmy (Callatini etal. (2005) Retrovirology. 2:30; Gessain and Mahieux. (2000) Bull. Soc.Pathol. Exot. 93:163-171; Meertens and Gessain. (2003) J. Virol.77:782-789; Meertens et al. (2002) J. Virol. 76:259-268; Meertens et al.(2003) J. Gen. Virol. 84:2723-2727; Takemura et al. (2002) J. Virol.76:1642-1648; Van Dooren et al. (2004) J. Gen. Virol. 85:507-519),collectively indicate that HTLV-3 infection be more frequent thanpreviously understood. In addition, the archaic age of the ancestralHTLVs and the recent finding of STLV-like infections in African hunterscollectively imply that cross-species transmission of STLVs to humans isboth an ancient and contemporary phenomenon coupled to behavior thatexposes humans to nonhuman primates. The ancient origin of HTLVcontrasts with that reported for HIV, which is believed to have onlycrossed over into humans from SIV-infected NHPs within the last century,and indicates a long period of viral evolution and adaptation in humanspossibly resulting in the observed lower pathogenicity for HTLV comparedto HIV (Hahn et al. (2000) Science 287:607-614; Sharp et al. (2000)Biochem Soc Trans. 28:275-282).

Screening for HTLV-3 can be facilitated by the application of diagnosticserologic and molecular assays based on the sequences reported here. Forexample, the data show that the Gag matrix and nucleocapsid regions andthe envelope surface protein are relatively conserved within PTLV-3 butare divergent from PTLV-1 and PTLV-2 and can thus be used todifferentiate the three PTLV groups with serological methods.

At the molecular level, examination of the genomic structure showed thatthe enzymatic, regulatory, and structural proteins are well preserved inHTLV-3(2026ND), including conserved functional motifs in Tax importantfor viral expression and T-cell proliferation. The finding of a PDZdomain in the Tax protein of HTLV-3(2026ND), like that seen in HTLV-1but not HTLV-2 (Feuer and Green. (2005) Oncogene. 24:5996-6004), whichhas been shown to be important in cellular signal transduction andT-cell transformation (Rousset et al. (1998) Oncogene. 6:643-654;Tsubata et al. (2005) Retrovirol. 2:46), indicates that the HTLV-3 Taxis more phenotypically similar to HTLV-1 than HTLV-2. The high aminoacid identity of the PTLV-3 Tax proteins combined with the ability ofSTLV-3 to transform human cells in vitro indicates that the HTLV-3 Taxfunctions similarly (Goubau et al. (1994) Proc. Natl. Acad. Sci. USA91:2848-2852).

In contrast to the tax gene, the HTLV-3(2026ND) LTR has only two of thethree conserved promoters identified in HTLV-1 and HTLV-2 that areresponsible for basal viral transcription levels and like STLV-3 ismissing the TATA-distal 21-bp repeat element (Meertens and Gessain.(2003) J. Virol. 77:782-789; Meertens et al. (2002) J. Virol.76:259-268; Meertens et al. (2003) J. Gen. Virol. 84:2723-2727; VanBrussel et al. (1997) J. Virol. 7:5464-5472; Van Dooren et al. (2004) J.Gen. Virol. 85:507-519). All of the remaining functional elements in theLTR were conserved, including the stem loop structure necessary for Rexresponsive control of viral expression in HTLV-1 and -2.

Recently, a HBZ protein was identified in translation of thecomplementary strand of the viral RNA genome between the env and tax/rexgenes (Gaudray et al. (2002) J Virol. 76:12813-12822). Proteintranslation on the minus-strand RNA is a unique feature of HTLV-1 notpreviously seen in retroviruses. HBZ was shown to be involved in thenegative regulation of viral replication (Gaudray et al. (2002) J Virol.76:12813-12822). The more recent finding of HBZ mRNA expression in ATLpatients indicates a role of HBZ mRNA in the survival of leukemic cellsin vivo and in HTLV-1-associated oncogenesis (Satou et al. (2006) Proc.Natl. Acad. Sci. USA. 103:720-725). Although originally reported to beexclusive to PTLV-1 (Gaudray et al. (2002) J Virol. 76:12813-12822), HBZis conserved among PTLVs, including HTLV-3(2026ND), demonstratingfurther the potential importance of this protein in viral replicationand oncogenesis. Of all PTLVs with full length genomes available atGenBank, only HTLV-2(MoT) did not have the full complement of leucineheptads in the leucine zipper due to a frameshift mutation in thepredicted HBZ sequence.

In summary, disclosed herein, HTLV-3(2026ND) is genetically stable andhas an ancient origin. HTLV-3(2026ND) genomic structure is relativelyconserved and contains many of the functional motifs important for theviral expression and pathology associated with HTLVs.

Materials and Methods

DNA Preparation and PCR-Based Genome Walking

DNA was prepared from uncultured PBMCs available from person 2026NDidentified in the original PTLV surveillance study in Cameroon reportedin detail elsewhere (Wolfe et al. (2005) Proc. Natl. Acad. Sci. USA.102:7994-7999). DNA integrity was confirmed by β-actin polymerase chainreaction (PCR) as previously described (Wolfe et al. (2005) Proc. Natl.Acad. Sci. USA. 102:7994-7999). All DNA preparation and PCR assays wereperformed in a laboratory where only human specimens are processed andtested according to recommended precautions to prevent contamination. Toobtain the full-length genomic sequence of HTLV-3 small regions of eachmajor coding region were PCR-amplified by using nested PCR anddegenerate PTLV primers. The tax (577-bp) and polymerase (pol) (709-bp)sequences were amplified by using primers and conditions providedelsewhere (Wolfe et al. (2005) Proc. Natl. Acad. Sci. USA.102:7994-7999). Envelope (env) (371-bp) sequences were amplified byusing standard PCR conditions with a 45° C. annealing temperature andthe external and internal primers PGENVF1 5′TGGATCCCGTGG(A/C)GI(C/T)TCCTIAA 3′ (SEQ ID NO: 27) and PGENVR1 5′GT(A/G)TAIG(C/G)(A/G)(C/G)AIGTCCAIG(A/C)(T/C)TGG 3′ (SEQ ID NO: 28) andPFENVF2 5′ AIAGACC(T/A)(C/T)CAAC(A/T)CCATGGGTAA 3′ (SEQ ID NO: 29) andPGENVR2 5′ G(A/C)(T/C)TGGCAICCIA(A/G)GTAIGGGCA 3′ (SEQ ID NO: 30),respectively. A 398-bp fragment of the long terminal repeat (LTR) wasobtained by using conserved STLV-3 primers as previously reported (Wolfeet al. (2005) Proc. Natl. Acad. Sci. USA. 102:7994-7999).

HTLV-3(2026ND)-specific primers were then designed from sequencesobtained in each of the four viral regions described above and were usedin nested, long-template PCRs to fill in the gaps in the genome asdepicted in FIG. 4 by using an expand high fidelity kit containing bothTaq and Tgo DNA polymerases (Roche). The external and internal primersequences for the LTR-pol and pol-env fragments are 2026LF1 5′GGTAAGATCCCACTGGGTCGAGC 3′ (SEQ ID NO: 69) and 2026PR1 5′GAAGCCAGGTCTCGGGTGACG 3′ (SEQ ID NO: 70) and 2026LF2 5′CGCTCCCCTGGAGCTCTCTCG 3′ (SEQ ID NO: 71) and 2026PR2 5′GCCACTTCCCATTGGGCTTTTTGACGG 3′ (SEQ ID NO: 72) and 2026 PF3 5′GCTCTCACCGATAAAGTAACAAACG 3′ (SEQ ID NO: 73) and 2026ER1 5′GGTAGGAAGAGGCTCCTATGAACAG 3′ (SEQ ID NO: 74) and 2026 PF2 5′CAGGACTGCATAACATACGAGACCCTCC 3′ (SEQ ID NO: 75) and 2026ER3 5′CCTATGAACAGGGTGCATCGACTGGG 3′ (SEQ ID NO: 76), respectively. Theexternal and internal primer sequences used to obtain about 3 kb of the3′ end of the genome (env-tax-LTR) are 2026EF1 5′ CCTAAGCCCCCCATGTCCAGAC3′ (SEQ ID NO: 77) and 2026LR1 5′ CGAGAGAGCTCCAGGGGAGCG 3′ (SEQ ID NO:78) and 2026EF3 5′ CCTACTCCCTGTATGTATTCCCCCATTGG 3′ (SEQ ID NO: 79) and2026LR2 5′ GCTCGACCCAGTGGGATCTTACCGAGTGG 3′ (SEQ ID NO: 80),respectively.

PCR products were revealed on 1.5% agarose gels stained with ethidiumbromide, purified with a Qiaquick PCR purification kit (Qiagen) andsequenced in both directions with a BigDye terminator cycle kit andautomated sequencers (Applied Biosystems). Selected PCR products werealso cloned into the pCR4-TOPO vector using the TOPO TA Cloning kit(Invitrogen) and recombinant plasmid DNA was prepared using the Qiagenplasmid purification kit prior to automated sequencing.

Sequence and Phylogenetic Analysis

Percent nucleotide divergence was calculated by using the GAP program inthe Genetic Computer Group's (GCG) Wisconsin package (Thompson et al.(1994) Nucleic Acids Res. 22:4673-4680). LTR RNA secondary structure wasdetermined using the program RNAstructure v4.2 (Mathews et al. (1999) J.Mol. Biol. 288:911-940). Sequences were aligned by using the Clustal Wprogram (Womble (2000) Methods Mol. Biol. 132:3-22), gaps were removed,and distance-based trees were generated by using the Kimuratwo-parameter model in conjunction with the neighbor-joining (NJ) methodin the MEGA program (version 2.1) and maximum likelihood (ML) analysisin the PAUP* program as described in detail elsewhere (Switzer et al.(2005) Nature. 434:376-380; Wolfe et al. (2005) Proc. Natl. Acad. Sci.USA. 102:7994-7999). The reliability of the final topology of the treeswas tested with 1,000 bootstrap replicates. Comparison of full-lengthPTLV genomes available at GenBank was done using HTLV-3(2026ND) as thequery sequence and the F84 (ML) model and a transition/transversionratio of 2.0 implemented in the program SimPlot (Lole et al. (1999) J.Virol. 73:152-160).

For dating of HTLV-3(2026ND), full-length genomes from prototypicalPTLVs available at GenBank were aligned with HTLV-3(2026ND) by usingClustal W, gaps were removed, and minor adjustments in the alignmentwere made manually. LTR sequences were excluded from the analysis sincethis region does not align accurately in PTLVs. The best fittingevolutionary model for the aligned sequences was determined withModeltest v3.6 (Posada and Crandall. (1998) Bioinformatics. 14:817-818).The general time-reversible model, allowing six different substitutionrate categories, with gamma-distributed rate heterogeneity (1.9724) andan estimated proportion of invariable sites (0.3687), was determined tobe the best fit to the data. Little substitution saturation was observedin the 7213-bp alignment (P<0.00001) as determined with the DAMBEprogram, and was therefore satisfactory for use in phylogeneticanalyses. Likewise, using the best-fitting evolutionary model definedabove, good phylogenetic signal in the alignment was also found withlikelihood mapping analysis using the program Tree-Puzzle v5.2.

The molecular clock hypothesis, or constant rate of evolution, wastested by using the likelihood ratio test with the likelihoods for theML and clock-like ML trees obtained in PAUP*. The clock was tested withthe best-fitting evolutionary model estimated in Modeltest, and ML treeswere constructed in PAUP* starting from the NJ tree that is iterativelyoptimized using two consecutive heuristic searches with nearest neighborinterchange followed by a final heuristic search with thetree-bisection-reconnection algorithm. To adjust for rate heterogeneityamong different PTLV taxa, clock-like ML trees were then transformedinto ultrametric trees using the nonparametric rate smoothing (NPRS)algorithm in the program TreeEdit (v1.0a10 carbon) (Sanderson (2003)Bioinformatics. 19:301-2). The branches of the NPRS tree were thenscaled by using a divergence time of 40,000-60,000 years ago (ya) forthe Melanesian HTLV-1mel lineage based on genetic and archaelogicalevidence of when the ancestors of indigenous Melanesians and Australiansmigrated from Southeast Asia (Lemey et al. (2005) Infect. Gen. Evol.5:291-298; Salemi et al. (2000) Mol. Biol. Evol. 17:374-386; Salemi etal. (1999) AIDS Rev. 1:131-139). Variance in age estimates (branchlengths) was determined in PAUP* with 100 bootstrap repetitions byenforcing topological constraints and using a heuristic search withoutbranch swapping on the clock-like ML tree. Branch lengths in all 100trees were calibrated as before and average divergence times andconfidence intervals (α=0.05) were calculated in Excel. The evolutionaryrate was estimated based on a known divergence time point of40,000-60,000 ya and on the branch length of the ML clock-like treeaccording to the formula: evolutionary rate (r)=branch length(bl)/divergence time (t) (Van Dooren et al. (2004) J. Gen. Virol.85:507-519).

Nucleotide Sequence Accession Number

The HTLV-3(2026ND) proviral sequence has the GenBank accession numberDQ093792.

Example 3 Generation and Analysis of the Human T-lymphotropic Virus Type4 Complete Genome

The full-length genomic sequence of HTLV-4 (SEQ ID NO: 81) shown in FIG.10 was obtained substantially as described above in Example 2 for theidentification if the HTLV-3 full-length genomic sequence. Briefly, DNAwas prepared from uncultured PBMCs available from a subject identifiedin the original PTLV surveillance study in Cameroon reported in detailelsewhere (Wolfe et al. (2005) Proc. Natl. Acad. Sci. USA.102:7994-7999). DNA integrity was confirmed by β-actin polymerase chainreaction (PCR) as previously described (Wolfe et al. (2005) Proc. Natl.Acad. Sci. USA. 102:7994-7999). To obtain the full-length genomicsequence of HTLV-3, small regions of each major coding region werePCR-amplified using nested PCR and degenerate PTLV primers.

HTLV-3(2026ND)-specific primers were then designed from sequencesobtained in each of the four viral regions described above (tax, pol,env, and LTR), and used in nested, long-template PCRs to fill in thegaps in the genome using an expand high fidelity kit containing both Taqand Tgo DNA polymerases (Roche). PCR products were revealed on 1.5%agarose gels stained with ethidium bromide, purified with a Qiaquick™PCR purification kit (Qiagen) and sequenced in both directions with aBigDye™ terminator cycle kit and automated sequencers (AppliedBiosystems). Selected PCR products were also cloned into the pCR4-TOPOvector using the TOPO TA Cloning kit (Invitrogen) and recombinantplasmid DNA was prepared using the Qiagen plasmid purification kit priorto automated sequencing.

Percent nucleotide divergence was calculated by using the GAP program inthe Genetic Computer Group's (GCG) Wisconsin package (Thompson et al.(1994) Nucleic Acids Res. 22:4673-4680). LTR RNA secondary structure wasdetermined using the program RNAstructure v4.2 (Mathews et al. (1999) J.Mol. Biol. 288:911-940). Sequences were aligned by using the Clustal Wprogram (Womble (2000) Methods Mol. Biol. 132:3-22), gaps were removed,and distance-based trees were generated by using the Kimuratwo-parameter model in conjunction with the neighbor-joining (NJ) methodin the MEGA program (version 2.1) and maximum likelihood (ML) analysisin the PAUP* program as described in detail elsewhere (Switzer et al.(2005) Nature. 434:376-380; Wolfe et al. (2005) Proc. Natl. Acad. Sci.USA. 102:7994-7999). The reliability of the final topology of the treeswas tested with 1,000 bootstrap replicates. Table 7 shows a comparisonof the genetic identity of the HTLV-3 and HTLV-4 full-length genomeswith other PTLV prototypes. The stem loop structure necessary for Rexresponsive control of viral expression in HTLV-1 and -2 was retained inHTLV-4(1863LE), and is shown in FIG. 11.

TABLE 7 Genetic Identity of HTLV-3 and HTLV-4 Genomes with other PTLVPrototypes (strain) HTLV-1 STLV-1 HTLV-2 STLV-2 STLV-3 STLV-3 HTLV-3HTLV-3 HTLV-4 (ATK) (Tan) (MoT) (PP1664) (TGE2117) (CTO604) (2026ND)(Pyl43) (1863LE) HTLV-3 2026ND 61.6 61.6 62.9 62.6 87.1 88.4 — 88.5 63.2Pyl43 62.3 62.1 63.1 63.2 87.7 99.2 88.5 — 63.0 HTLV-4 1863LE 62.0 62.070.7 70.8 63.5 63.1 63.2 63.0 —

For dating of HTLV-4(1863LE), full-length genomes from prototypicalPTLVs available in GenBank were aligned with HTLV-3(2026ND) andHTLV-4(1863LE) essentially as described in Example 2 using Clustal W.The analysis, shown in FIG. 12, again inferred four major phylogroupswith very high bootstrap support, confirming the genetic relationshipsthat were based on smaller sequences. Both HTLV-3s again clustered withSTLV-3s supporting a primate origin for these viruses. HTLV-4 againformed a new lineage distinct from PTLV-1, PTLV-2, and PTLV-3. However,the primate origin of HTLV-4 was less clear since there is not yet aknown simian counterpart for this virus. These results also indicatedthe absence of genetic recombination in PTLVs, which is a commonmechanism that leads to increased genetic diversity of HIV.

The finding of HTLVs in four distinct clades indicated an ancient,independent evolution of these viruses. Thus, additional molecularanalyses were performed to estimate the divergence times of the PTLVlineages. FIG. 13 shows the estimated divergence dates for the mostrecent common ancestor of HTLV-3(2026ND), HTLV-4(1863LE) and otherPTLVs. Using the bovine leukemia virus (BLV) as an outgroup, asubstitution rate of 8.6×10⁻⁷ to 1.3×10⁻⁶ substitutions/site/year forPTLV was inferred which is 3 logs lower than that seen in HIV,confirming the genetic stability of these deltaretroviruses.

Using these substitution rates, molecular dating inferred an ancientorigin for PTLVs hundreds of thousands of years ago with the most recentcommon ancestor for each HTLV group ranging from 30,000 years ago forHTLV-2 to 456,000 years ago for HTLV-4. This finding contrasts with themore recent origin of HIV-1, which has been estimated to have occurredwithin the last century.

The inferred ancient origin for HTLV-3 and HTLV-4 indicates thatexposure to these viruses may have been occurring for millennia, andthus these viruses may be more prevalent than currently known.Alternatively, HTLV-3 and HTLV-4 may represent more recent infectionswith highly divergent STLVs that have yet to be identified. This isprobably the case for the HTLV-3(Py143) strain, since the high geneticidentity of this virus to STLV-3RCM is similar to that seen intransmission pairs. Expanded surveillance of both humans and primates iswarranted.

Changes in the molecular structure and genetic sequences of viruses hasbeen proposed to play a role in the increased transmissibility andpathogenicity of viruses following cross-species transmission andadaptation to a new host. Thus, the genetic structure and sequences ofHTLV-3 and HTLV-4 were examined to determine if the genome was intactand if important functional motifs involved in viral expression andHTLV-1-induced leukemogenesis are preserved. The Tax proteins of HTLV-3were also characterized using in vitro assays to determine if motifsinvolved in Tax-mediated leukemogenesis were present and functioning.

While all structural and enzymatic proteins of both HTLV-3s and HTLV-4were intact, features that are either unique or similar to those ofother HTLVs were identified (see Table 8). First, the genomes of HTLV-3and HTLV-4 are shorter than HTLV-1 and HTLV-2 by having only two ofthree Tax response elements in the LTRs. However, the loss of thisdistal TRE has been shown to not significantly affect HTLV expression.In addition, only two TREs are present in STLV-3 and STLV-2 suggestingthis difference is not a result of adaptation to a new host. Likewise,the finding of AP-1 and c-Myb transcription factors in place of theHTLV-3 or HTLV-4 LTRs is also not unique but are also present in STLV-3.

Overall, the HTLV-3 Tax protein contains many of the functional motifsimportant for viral expression and leukemogenesis attributed to HTLV-1Tax. Detailed in vitro analysis confirmed that the HTLV-3 Tax wassimilar in function to the HTLV-1 Tax protein, suggesting a pathogenicpotential in HTLV-3-infected persons like that observed in HTLV-1. TheHTLV-3(Py143) genome is also shorter by a 366-bp deletion in the pXregion that disrupts the HBZ reading frame suggesting a loss of Taxsuppression and T-cell proliferation believed to be associated with thisgene.

TABLE 8 Unique Genetic Features of HTLV Prototypes: HTLV-3 is moresimilar to HTLV-1 HTLV-1 HTLV-2 HTLV-3 HTLV-3 HTLV-4 (ATK) (MoT)(2026ND)* (Pyl43)** (1863LE) Genome (bp) 9068 8952 8917 8553 8791 LTR(bp) 756 764 697 695 696 # LTR TREs 3 3 2¹ 2¹ 2¹ Other LTR TFs — — AP-1c-Myb c-Myb Tax transactivates Yes Yes Yes Yes Tax localization NucleusCytoplasm Nucleus Tax p53 inhibition Yes Yes PDZ BD in Tax Yes No  YesYes No HBZ Yes No² Yes No³ Yes ¹missing distal TRE ²HBZ is present inother HTLV-2 ³366-bp deletion in pX *Switzer et al. J Virol. 2006; 80:7427-38. **Calattini et al. J Virol. 2006; 80: 9876-88.

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SEQUENCES

-   SEQ ID NO: 1 (HTLV-3 pol) (pos 2407-5076)-   SEQ ID NO: 2 (HTLV-4 pol) (pos 3-2549)-   SEQ ID NO: 3 (HTLV-3 env) (pos 5069-6544)-   SEQ ID NO: 4 (HTLV-4 env) (pos 2542-3999)-   SEQ ID NO: 5 (HTLV-3 tax)-   SEQ ID NO: 6 (HTLV-4 tax)-   SEQ ID NO: 7 (PTLVTPG)-   SEQ ID NO: 8 (PGTAXR1)-   SEQ ID NO: 9 (PH2Rrev)-   SEQ ID NO: 10 (PGTAXR2)-   SEQ ID NO: 11 (PGPOLF1)-   SEQ ID NO: 12 (PGPOLR1)-   SEQ ID NO: 13 (PGPOLF2)-   SEQ ID NO: 14 (PGPOLR2)-   SEQ ID NO: 15 (1863TF1)-   SEQ ID NO: 16 (1863TR1)-   SEQ ID NO: 17 (1863TF2)-   SEQ ID NO: 18 (1863TR2)-   SEQ ID NO: 19 (5VLTRext)-   SEQ ID NO: 20 (1MNDR1)-   SEQ ID NO: 21 (Enh280)-   SEQ ID NO: 22 (1MNDR2)-   SEQ ID NO: 23 (PH1F)-   SEQ ID NO: 24 (PH2R)-   SEQ ID NO: 25 (PH2F)-   SEQ ID NO: 26 (PH2R)-   SEQ ID NO: 27 (PGENVF1)-   SEQ ID NO: 28 (PGENVR1)-   SEQ ID NO: 29 (PGENVF2)-   SEQ ID NO: 30 (PGENVR2)-   SEQ ID NO: 31 (GPLTRF1)-   SEQ ID NO: 32 (GPLTRR1)-   SEQ ID NO: 33 (GPLTRF2)-   SEQ ID NO: 34 (GPLTRR2)-   SEQ ID NO: 35 (HTLV-3 gag) (pos 756-2023)-   SEQ ID NO: 36 (HTLV-3 Complete genome: 2026ND.seq (8917 bp)-   SEQ ID NO: 37 (HTLV-3 env amino acid)-   SEQ ID NO: 38 (HTLV-3 env surface antigen (SU)=aa 1-315)-   SEQ ID NO: 39 (HTLV-3 env transmembrane=aa 316-491)-   SEQ ID NO: 40 (HTLV-3 gag amino acid)-   SEQ ID NO: 41 (HTLV-3 gag p15=aa 337-422)-   SEQ ID NO: 42 (HTLV-3 gag p19=aa 1-123)-   SEQ ID NO: 43 (HTLV-3 gag p24=aa 124-336)-   SEQ ID NO: 44 (HTLV-3 pol) amino acid-   SEQ ID NO: 45 (HTLV-3 LTR) (pos 1-697 & 8221-8917)-   SEQ ID NO: 46 (HTLV-3 pro) amino acid-   SEQ ID NO: 47 (HTLV-3 pro) (pos 1976-2509)-   SEQ ID NO: 48 (HTLV-3 rex) amino acid-   SEQ ID NO: 49 (HTLV-3 rex)(pos 5010-5071..7245-7730)-   SEQ ID NO: 50 (HTLV-3 tax) amino acid-   SEQ ID NO: 51 (HTLV-3 tax) (pos5069-5071..7244-8293)-   SEQ ID NO: 52 (HTLV-3 pX (pos 6545-7243)-   SEQ ID NO: 53 (HTLV-4 pol-env-tax region)-   SEQ ID NO: 54 (HTLV-4 env) amino acid-   SEQ ID NO: 55 (HTLV-4 env surface antigen (SU)=aa 1-307)-   SEQ ID NO: 56 (HTLV-4 env transmembrane=aa 308-485)-   SEQ ID NO: 57 (HTLV-4 pol) amino acid-   SEQ ID NO: 58 (HTLV-4 pro) amino acid-   SEQ ID NO: 59 (HTLV-4 pro) (pos 1-273)-   SEQ ID NO: 60 (HTLV-4 rex) amino acid-   SEQ ID NO: 61 (HTLV-4 rex) (pos 2483-2545..4560-5009)-   SEQ ID NO: 62 (HTLV-4 tax) amino acid-   SEQ ID NO: 63 (HTLV-4 pX) (pos.4000-4558)-   SEQ ID NO: 64 1863 PF1-   SEQ ID NO: 65 1863PR2-   SEQ ID NO: 66 1863PP2 FAM (flourescent labeled probe)-   SEQ ID NO: 67 region of HTLV-3 where type specific peptides of    HTLV-1 and HTLV-2 are located-   SEQ ID NO: 68 region of HTLV-4 where type specific peptides of    HTLV-1 and HTLV-2 are located-   SEQ ID NO: 69 2026LF1-   SEQ ID NO: 70 2026PR1-   SEQ ID NO: 71 2026LF2-   SEQ ID NO: 72 2026PR2-   SEQ ID NO: 73 2026 PF3-   SEQ ID NO: 74 2026ER1-   SEQ ID NO: 75 2026 PF2-   SEQ ID NO: 76 2026ER3-   SEQ ID NO: 77 2026EF1-   SEQ ID NO: 78 2026LR1-   SEQ ID NO: 79 2026EF3-   SEQ ID NO: 80 2026LR2-   SEQ ID NO: 81 (HTLV-4(1863LE) Complete genome)-   SEQ ID NO: 82 (GTTAAAC)-   SEQ ID NO: 83 (TTTAAAC)-   SEQ ID NO: 84 HTLV-3 HBZ

1. An isolated primate T-lymphotropic virus comprising: (a) a gag genecomprising the nucleic acid sequence of SEQ ID NO: 35; a pol genecomprising a nucleic acid sequence at least 92.8% identical to SEQ IDNO: 1; and an env gene comprising a nucleic acid sequence at least 92.5%identical to SEQ ID NO: 3; or (b) a gag gene comprising the nucleic acidsequence of nucleotides 750-2024 of SEQ ID NO: 81: a pol gene comprisinga nucleic acid sequence at least 71.5% identical to SEQ ID NO: 2; and anenv gene comprising a nucleic acid sequence at least 73.5% identical toSEQ ID NO:
 4. 2. A vector comprising the isolated primate T-lymphotropicvirus of claim
 1. 3. The vector of claim 2, further comprising aheterologous nucleic acid.
 4. The vector of claim 3, wherein theheterologous nucleic acid is a nucleic acid encoding an antigen from aknown pathogen, or a cancer-related antigen, under the control of apromoter element.
 5. The vector of claim 4, wherein the pathogencomprises: a virus selected from the group of viruses consisting ofHerpes simplex virus type-1, Herpes simplex virus type-2,Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Humanherpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Variola virus,Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus,Coronavirus, Influenza virus A, Influenza virus B, Measles virus,Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, SARS,Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus,Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus,Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus,Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valleyfever virus, West Nile virus, Rift Valley fever virus, Rotavirus A,Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus,Human T-lymphotropic virus type-1, Human T-lymphotropic virus type-2,Primate T-lymphotropic virus, Hantavirus, Rubella virus, HumanImmunodeficiency virus type-1, Human Immunodeficiency virus type-2, andSimian Immunodeficiency virus (SIV).
 6. The vector of claim 4, whereinthe cancer-related antigen comprises: a cancer-related antigen selectedfrom the group of cancers consisting of lymphoma, B cell lymphoma, Tcell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia,bladder cancer, brain cancer, nervous system cancer, head and neckcancer, squamous cell carcinoma of head and neck, kidney cancer, lungcancers such as small cell lung cancer and non-small cell lung cancer,neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostatecancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas ofthe mouth, throat, larynx, and lung, colon cancer, cervical cancer,cervical carcinoma, breast cancer, and epithelial cancer, renal cancer,genitourinary cancer, pulmonary cancer, esophageal carcinoma, head andneck carcinoma, large bowel cancer, hematopoietic cancers; testicularcancer; colon and rectal cancers, prostatic cancer, and pancreaticcancer.
 7. A method of eliciting an immune response in a subject againstan illness associated with a pathogen, comprising administering thevector of claim
 2. 8. The vector of claim 4, wherein the pathogen is abacterium selected from the group of bacteria consisting of M.tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M. avium,M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans,M. avium subspecies paratuberculosis, Nocardia asteroides, otherNocardia species, Legionella pneumophila, other Legionella species,Salmonella typhi, other Salmonella species, Shigella species, Yersiniapestis, Pasteurella haemolytica, Pasteurella multocida, otherPasteurella species, Actinobacillus pleuropneumoniae, Listeriamonocytogenes, Listeria ivanovii, Brucella abortus, other Brucellaspecies, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydiatrachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsialspecies, Ehrlichia species, Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillusanthracis, Escherichia coli, Vibrio cholerae, Campylobacter species,Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi,other Hemophilus species, Clostridium tetani, other Clostridium species,Yersinia enterolitica, and other Yersinia species.
 9. The vector ofclaim 4, wherein the pathogen is a parasite selected from the group ofparasites consisting of Toxoplasma gondii, Plasmodium falciparum,Plasmodium vivax, Plasmodium malariae, other Plasmodium species.,Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, otherLeishmania species., Schistosoma mansoni, other Schistosoma species.,and Entamoeba histolytica.
 10. The vector of claim 4, wherein thepathogen is a fungus selected from the group of fungi consisting ofCandida albicans, Cryptococcus neoformans, Histoplama capsulatum,Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodesbrasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicilliummarneffi, and Alternaria alternata.
 11. The vector of claim 6, whereinthe cancer related antigen is selected from the group consisting ofhuman epithelial cell mucin, the Ha-ras oncogene product, p53,carcino-embryonic antigen (CEA), the raf oncogene product, gp100/pmel17,GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, gp75,Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7,prostate-specific antigen (PSA), HPV-16, MUM, alpha-fetoprotein (AFP),CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7, Wilm'stumor antigen-1, telomerase, and melanoma gangliosides.
 12. An isolatedprimate T-lymphotropic virus comprising a nucleic acid sequence with atleast 98% identity to SEQ ID NO: 36 or SEQ ID NO:
 81. 13. The isolatedprimate T-lymphotropic virus of claim 12, comprising the nucleic acidsequence of SEQ ID NO: 36 or SEQ ID NO:
 81. 14. A vector comprising theisolated primate T-lymphotropic virus of claim
 12. 15. The vector ofclaim 14, further comprising a heterologous nucleic acid.
 16. A methodof eliciting an immune response in a subject against an illnessassociated with a pathogen, comprising administering to the subject thevector of claim
 15. 17. The isolated primate T-lymphotropic virus ofclaim 13, wherein the nucleic acid sequence of the isolated primateT-lymphotropic virus consists of SEQ ID NO: 36 or SEQ ID NO:
 81. 18. Theisolated primate T-lymphotropic virus of claim 1, comprising: a gag genecomprising the nucleic acid sequence of SEQ ID NO: 35; a pol genecomprising the nucleic acid sequence of SEQ ID NO: 1; and an env genecomprising the nucleic acid sequence of SEQ ID NO:
 3. 19. The isolatedprimate T-lymphotropic virus of claim 18, further comprising: (a) a taxgene comprising the nucleic acid sequence of SEQ ID NO: 5; (b) a rexgene comprising the nucleic acid sequence of SEQ ID NO: 49; (c) LTRcomprising the nucleic acid sequence of SEQ ID NO: 45; or (d) two ormore of (a), (b) and (c).
 20. The isolated primate T-lymphotropic virusof claim 1, comprising: a gag gene comprising the nucleic acid sequenceof nucleotides 750-2024 of SEQ ID NO: 81; a pol gene comprising thenucleic acid sequence of SEQ ID NO: 2; and an env gene comprising thenucleic acid sequence of SEQ ID NO:
 4. 21. The isolated primateT-lymphotropic virus of claim 20, further comprising: (a) a tax genecomprising the nucleic acid sequence of SEQ ID NO: 6; (b) a rex genecomprising the nucleic acid sequence of SEQ ID NO: 61; (c) LTRcomprising the nucleic acid sequence of nucleotides 1-749 and 6560-8791of SEQ ID NO: 81; or (d) two or more of (a), (b) and (c).
 22. Anisolated primate T-lymphotropic virus comprising: a gag gene comprisingthe nucleic acid sequence of SEQ ID NO: 35; a pol gene comprising anucleic acid sequence at least 92.8% identical to SEQ ID NO: 1; and anenv gene comprising a nucleic acid sequence at least 92.5% identical toSEQ ID NO:
 3. 23. An isolated primate T-lymphotropic virus comprising: agag gene comprising the nucleic acid sequence of nucleotides 750-2024 ofSEQ ID NO: 81; a pol gene comprising a nucleic acid sequence at least71.5% identical to SEQ ID NO: 2; and an env gene comprising a nucleicacid sequence at least 73.5% identical to SEQ ID NO: 4.